JP5685712B2 - Apoptosis detection method - Google Patents

Description

  The present invention relates to a method for detecting apoptosis, an apoptosis detection agent, an apoptosis detection kit, and a screening method for candidate substances for apoptosis inducers or apoptosis inhibitors.

The abbreviations used in the application documents and their significance are as follows.
2SH: (6-O · N) -desulfated / N-acetylated HEP
6DSH: 6-O-desulfated HEP
6SH: (2-O · N) -desulfated / N-acetylated HEP
AS: Acalane sulfate ACH: 2-O-desulfated AS
Ac-6DSH: N-acetylated 6DSH
Ac-NAH: N-acetylated NAH
Ac-NSH; N-acetylated NSH
CDSH: Completely desulfated / N-acetylated HEP
Ch: Chondroitin CS: Chondroitin sulfate CS-A (S): Shark-derived chondroitin sulfate A
CS-A (W): Whale-derived chondroitin sulfate A
CS-B: Chondroitin sulfate B (synonymous with DS)
CS-C: Chondroitin sulfate C
CS-D: Chondroitin sulfate D
CS-E: Chondroitin sulfate E
DMEM: Dulbecco's modified Eagle medium DS: Dermatan sulfate (synonymous with CS-B)
DTT: Dithiothreitol EHS-HS: HS from mouse Angelbreth-Holm-Swarm sarcoma
ELISA method: Enzyme-labeled antibody measurement method FCS: Fetal calf serum FITC: Fluorescein isothiocyanate G3PDH: Glyceraldehyde-3-phosphate dehydrogenase GlcA: Glucuronic acid GlcN: N-acetylglucosamine GlcNAc: N-acetylglucosamine GlcNS : N-sulfated glucosamine HA: hyaluronic acid HEP: heparin HS: heparan sulfate HS2ST: HS 2-O-sulfotransferase HS6ST: HS 6-O-sulfotransferase IdoA: iduronic acid IdoA (2S): 2- O-sulfated iduronic acid KS: keratan sulfate NAc-HEP: N-desulfated / N-acetylated HEP
NAH: N-acetylheparosan NDST: HS / HEP GlcNAc N-deacetyl / GlcN N-sulfotransferase (heparan sulfate / heparin GlcNAc N-deacetylase / GlcN N-sulfotransferase)
NH ₂ -2SH: (6-O · N) -desulfated HEP
NH ₂ -6SH: (2-O · N) -desulfated HEP
NH ₂ -CDSH: Completely desulfated HEP
NH ₂ -HEP: N-desulfated HEP
NSH: (2-O · 6-O) -desulfated HEP
PBS: phosphate buffered saline PDNAc-NAH: partially deacetylated NAH

It is known that apoptosis can be detected by using chromosome aggregation of cell nuclei, fragmentation of cell nuclei, chromosomal DNA ladder, etc. as an index (Non-patent Document 1). A method for detecting apoptosis using an antibody that recognizes a Le ^Y sugar chain is also known (Patent Document 1).
However, there is no known method for detecting apoptosis using glycosaminoglycan or proteoglycan as an index.
On the other hand, for example, JM403 antibody (Patent Document 2) and NAH43 antibody (Patent Document 3) are known as antibodies that recognize and bind to GlcN residues in the HEP skeleton and HS skeleton. For example, the JM403 antibody is known to detect lysosomal disease (Patent Document 2), and the NAH43 antibody is known to detect HS or NAH (Patent Document 3). However, none of these antibodies are known for use as apoptotic detection applications.
Further, NDST-1, -2, -3, and -4 are known as enzymes involved in the biosynthesis of HEP and HS (Non-patent Document 2), but apoptosis is detected using the expression of these enzymes as an index. Also not known to do.

  In the early stage of apoptosis, the cell surface changes and translocation of phosphatidylserine from the inner side of the plasma membrane to the outer layer occurs. Therefore, this translocation is performed using annexin V that binds to phosphatidylserine with high affinity. A technique for detecting apoptosis by detecting a seated phosphatidylserine is also known. However, since annexin V is calcium-dependent and the presence of calcium has a significant effect on the detection results, this technique is capable of adhering cells (calculation requires the use of calcium to remove cells from the culture dish for analysis). Cannot be used.

JP-A-6-109729 JP 2005-524074 A International Publication No. 2006/106950 Pamphlet

Supervised by Kazutomo Imahori and Tamio Yamakawa, “Biochemical Dictionary” (3rd edition), Tokyo Chemical Co., Ltd., published on October 8, 1998, p54-55 Aikawa, Jun-ichi et al., 2001, The Journal of Biological Chemistry, Vol. 276, No. 8, p. 5876-5882

  An object of the present invention is to provide a novel method for detecting apoptosis, an apoptosis detection agent, an apoptosis detection kit, and a screening method for candidate substances for apoptosis inducers or apoptosis inhibitors.

  As a result of intensive studies to solve the above problems, the present inventors have found that apoptosis can be detected by detecting an increase in GlcN residues in the HEP skeleton or HS skeleton present in cells. Furthermore, it has been found that apoptosis can be detected by detecting an increase in the expression of at least one of NDST-3 and NDST-4 in cells. Furthermore, based on these findings, the present inventors have provided an apoptosis detection agent, an apoptosis detection kit, and a screening method for candidate substances for apoptosis inducers or apoptosis inhibitors.

That is, the present invention provides a method for detecting apoptosis (hereinafter referred to as “detection method 1 of the present invention”) including at least a step of detecting an increase in GlcN residues in the HEP skeleton or HS skeleton present in cells.
The GlcN residue is preferably present as a structure having a glycosidic bond with the GlcA residue.
In addition, this detection is preferably performed by an antibody. The antibody used in this case is preferably an antibody that binds to a GlcN residue in the HEP skeleton or HS skeleton.

The present invention also provides an apoptosis detection agent (hereinafter referred to as “the detection agent of the present invention”) comprising as an active ingredient an antibody that binds to a GlcN residue in the HEP skeleton or HS skeleton.
The GlcN residue to which this antibody binds preferably has a glycosidic bond with the GlcA residue.

The present invention also provides an apoptosis detection kit (hereinafter referred to as “the kit of the present invention”) comprising at least the components shown in the following (1) and (2).
(1) an antibody that binds to a GlcN residue in the HEP skeleton or HS skeleton,
(2) A reagent for detecting the antibody of (1) above.
The GlcN residue to which this antibody binds preferably has a glycosidic bond with the GlcA residue.

The present invention also comprises an apoptosis inducer or apoptosis inhibitor comprising at least a step of contacting a test substance with a cell and selecting the test substance when the GlcN residue in the HEP skeleton or HS skeleton in the cell is increased or decreased. A screening method for drug candidate substances (hereinafter referred to as “the present screening method 1”) is provided.
The GlcN residue is preferably present as a structure having a glycosidic bond with the GlcA residue.

  The present invention also provides a method for detecting apoptosis (hereinafter referred to as “detection method 2 of the present invention”) comprising at least a step of detecting an increase in expression of at least one of NDST-3 and NDST-4 in a cell. In particular, this “expression” is preferably the expression of mRNA.

  The present invention also includes an apoptosis-inducing agent comprising at least a step of contacting a test substance with a cell and selecting the test substance when the expression of at least one of NDST-3 and NDST-4 in the cell increases or decreases. Alternatively, a screening method for a candidate substance for an apoptosis inhibitor (hereinafter referred to as “the present screening method 2”) is provided. In particular, this “expression” is preferably the expression of mRNA.

  Hereinafter, the detection methods 1 and 2 of the present invention are collectively referred to as “the detection method of the present invention”. Further, the screening methods 1 and 2 of the present invention are collectively referred to as “the screening method of the present invention”.

  The detection method of the present invention is extremely useful because it enables objective detection of apoptosis using an index qualitatively different from conventional methods for detecting apoptosis (apoptotic body detection and DNA fragmentation). It is. In addition, the detection agent and the kit of the present invention are extremely useful because they can make the detection method of the present invention easier and faster. Furthermore, the screening method of the present invention is extremely useful because it can easily and rapidly screen for an apoptosis inducer or apoptosis inhibitor candidate substance that can also be used as a drug candidate substance.

It is a figure (photograph) which shows the agarose electrophoresis of the chromosomal DNA of K562 strain | stump | stock processed for 2 days with daunomycin of 1 micromol of final concentration. It is a figure which shows the grade of the coupling | bonding of various antibodies to K562 strain | stump | stock. A to E are cells not treated with daunomycin, F to J are cells treated with daunomycin at a final concentration of 2 μM for 2 days, and K to O are results of cells treated with daunomycin at a final concentration of 5 μM for 2 days. is there. It is a figure which shows the grade of the coupling | bonding of various antibodies to HL60 strain | stump | stock. A to C are results of cells not treated with daunomycin, and D to F are results of cells treated with daunomycin at a final concentration of 3 μM for 2 days. It is a figure which shows the grade of the coupling | bonding of various antibodies to U937 strain. A to C are results of culturing cells for 4 days in the presence of 10% FCS, and D to F are results of treating cells for 4 days under serum-deficient conditions (in the presence of 0.6% FCS). It is a figure which shows the grade of the coupling | bonding of various antibodies to U937 strain. A to C are results of cells not treated with daunomycin, and D to F are results of cells treated with daunomycin at a final concentration of 0.2 μM for 2 days. It is a figure which shows the grade of the coupling | bonding of various antibodies to C-1 stock | strain. All are the results of culturing cells in the presence of 10% FCS for 4 days. It is a figure which shows the grade of the coupling | bonding of various antibodies to C-1 stock | strain. All are the results of culturing cells for 4 days under serum-deficient conditions (in the presence of 0.6% FCS). It is a figure which shows the grade of the coupling | bonding of the various antibodies to LS174T strain. All are the results of culturing cells in the presence of 10% FCS for 4 days. It is a figure which shows the grade of the coupling | bonding of the various antibodies to LS174T strain. All are the results of culturing cells for 4 days under serum-deficient conditions (in the presence of 0.6% FCS). It is a figure which shows the grade of the coupling | bonding of the various antibodies to PC12 stock | strain. A to C are results of culturing cells for 5 days in the presence of 10% FCS, and D to F are results of treating cells for 5 days under serum-deficient conditions (in the presence of 0.6% FCS). It is a figure which shows the grade of the coupling | bonding of the various antibodies to PC12 stock | strain. A to C are results of cells not treated with daunomycin, and D to F are results of cells treated with daunomycin at a final concentration of 1 μM for 2 days. It is a figure which shows the grade of the coupling | bonding of various antibodies to a Jurkat strain | stump | stock. A to C are results of culturing cells for 5 days in the presence of 10% FCS, and D to F are results of treating cells for 4 days under serum-deficient conditions (in the presence of 0.6% FCS). It is a figure which shows the grade of the coupling | bonding of various antibodies to a Jurkat strain | stump | stock. A to C are results of cells not treated with daunomycin, and D to F are results of cells treated with daunomycin at a final concentration of 1 μM for 2 days. The figure (photograph) which shows the change of mRNA expression of various NDST by processing K562 strain with daunomycin.

<1> Detection method 1 of the present invention
The detection method 1 of the present invention is an apoptosis detection method including at least a step of detecting an increase in GlcN residues in a HEP skeleton or HS skeleton present in a cell.
The “cell” to be detected by apoptosis according to the detection method 1 of the present invention is not particularly limited as long as it is a cell that can cause apoptosis, and a cell for which apoptosis detection is desired may be used as it is. Examples of cells include cancer cells, blood cells such as T cells, nerve cells, and the like. For example, if it is desired to detect apoptosis in human cancer cells, the cancer cells may be used as they are.

The detection method 1 of the present invention includes at least a step of detecting an increase in GlcN residues in the HEP skeleton or HS skeleton present in such a cell. The GlcN residue in the present application document is preferably “a glucosamine residue in which the N-position is not substituted with either an acetyl group or a sulfate group”.
In addition, the GlcN residue in the present application document preferably exists as a structure in which a GlcA residue and a glycoside bond are present.
Therefore, the most preferable GlcN residue in the present application document is a “glucosamine residue in which the N-position is not substituted with either an acetyl group or a sulfate group”, and this is a structure in which a glycoside bond is formed with a GlcA residue. It exists.

  The method for detecting a GlcN residue in a HEP skeleton or HS skeleton present in a cell is not particularly limited as long as such a method capable of detecting a specific sugar chain structure is used. For example, the sugar chain of a cell to be detected for apoptosis may be extracted, and the GlcN residue in the HEP skeleton or HS skeleton may be detected from the extracted sugar chain by a known physicochemical analysis technique.

The sugar chain structure can also be detected by an antigen-antibody reaction using an antibody that binds to a GlcN residue in the HEP skeleton or HS skeleton.
The antibody used in this case is not particularly limited as long as it binds to a GlcN residue in the HEP skeleton or HS skeleton. Especially, it is preferable that the antigen binding property disappears when the antigen to which the antibody binds is treated with heparitinase. Moreover, what does not couple | bond with substantially any of HA, CS, DS, and KS is preferable. That is, those that specifically bind to the sugar chain structure are preferred.
The antibody may be either a monoclonal antibody or a polyclonal antibody, but is preferably a monoclonal antibody from the viewpoint of ensuring continuous productivity and antibody homogeneity. Further, in the present application document, “substantially does not bind” does not mean that one molecule does not bind, and it is weak even if the binding cannot be detected or the binding can be detected. Means negligible for those skilled in the art.

Specific antibodies that can be used in the detection method 1 of the present invention and are generally available to those skilled in the art include, for example, the JM403 antibody (Japanese Patent Publication No. 2005-524074: Diabetologia, 37 (3), p313-320 (1994). )). This antibody is commercially available from Seikagaku Biobusiness Corporation and is generally available. The JM403 antibody is a monoclonal antibody whose immunoglobulin class is mouse IgM and binds to a GlcN residue present in the HEP or HS backbone.
Further, when the antigen is treated with heparitinase, the binding property is lost, and it does not substantially bind to HA, CS, DS, or KS.
An antibody having the same complementarity determining region (CDR) as the JM403 antibody can also be used in the present invention.

Another specific antibody that can be used in the detection method 1 of the present invention and generally available to those skilled in the art includes, for example, the NAH43 antibody (WO 2006/106950 pamphlet). This antibody was released on March 11, 2005 by the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (currently the National Institute of Technology and Evaluation).
Produced by a hybridoma deposited under the accession number FERM BP-10535 in the Patent Organism Depositary Center, 2-5, Kazusa-Kamashita, Kisarazu City, Chiba, Japan, Room 8 120, Postal Code 292-0818). The NAH43 antibody is a monoclonal antibody, and its immunoglobulin class is mouse IgM, which binds to a GlcN residue present in the HEP or HS backbone. On the other hand, it does not substantially bind to EHS-HS, HA, Ch, various CS (CS-A, CS-B, CS-C, CS-D, CS-E) and KS.
An antibody having the same complementarity determining region (CDR) as the NAH43 antibody can also be used in the present invention.

  The antibody that can be used here can also be produced by a known method using, as an antigen, a GlcN residue in the HEP skeleton or HS skeleton, preferably a GlcN residue glycosidically bound to a GlcA residue. For example, if a monoclonal antibody is desired, a mammal is immunized with the antigen, lymphocytes are collected from the animal, and a hybridoma is formed by cell fusion with a myeloma cell derived from a mammal. A hybridoma that produces an antibody that specifically binds to the antigen may be selected and the antibody collected.

The antibodies that can be used here may be purified as immunoglobulins or may be unpurified (eg, hybridoma culture supernatant, ascites, antiserum itself, etc.) Those purified as immunoglobulins are preferred. In this application document, the term “purification” means not only so-called complete purification (purification until substantially pure), but also partial purification (not necessarily substantially pure, It is also used as a concept that includes purification to such an extent that the ratio of the target substance to be purified becomes large.
In addition, antibodies that can be used here are treated with a protease (for example, plasmin, pepsin, papain, etc.) that does not degrade the antigen binding site (Fab) as well as those that completely retain the molecular structure of immunoglobulin. It may be a fragment containing Fab. Examples of the fragment containing Fab of the antibody include Fabc, (Fab ′) _{2 and the} like in addition to Fab.

  In addition, if the nucleotide sequences of the genes encoding the antibodies that can be used here and the amino acid sequences of these antibodies are determined, these antibodies, Fab-containing fragments, chimeric antibodies, etc. may be prepared by genetic engineering. it can. Fragments including antibody Fabs, chimeric antibodies, and the like are also included in the concept of “antibody” in the present application document as long as they bind to the sugar chain structure.

  When detecting the sugar chain structure using an antibody, for example, the antibody may be brought into contact with a sugar chain extracted from a cell to be detected for apoptosis to cause an antigen-antibody reaction. The antibody may be directly contacted to cause an antigen-antibody reaction. In the former case, the sugar chain structure can be detected by, for example, immunoblotting, and in the latter case, the sugar chain structure can be detected by, for example, flow cytometry. In addition, about the method and conditions, etc. which detect an antigen (the said sugar chain structure) by making an antibody contact and make an antigen antibody reaction, the method and conditions of a general immunoassay can be utilized as it is.

The antibody that can be used here may be one directly bound to a labeling substance. Alternatively, an antibody that can be used here may be a primary antibody, and a secondary antibody that binds to the primary antibody and has a labeling substance bound thereto (an antibody that binds to an immunoglobulin related to the primary antibody) may be used.
As the labeling substance, for example, an enzyme (peroxidase, alkaline phosphatase, beta-galactosidase, luciferase, acetylcholine esterase, glucose oxidase and the like), radioisotopes ^{(125 I, 131 I, 3} H , etc.), fluorescent dyes (FITC, 7-
Amino-4-methylcoumarin-3-acetic acid (AMCA), dichlorotriazinylaminofluorescein (DTAF), tetramethylrhodamine isothiocyanate (TRITC), lithamine rhodamine B (Lissamine Rhodamine B), Texas Red, Texas Red, Phycoerythrin (PE), umbelliferone, europium, phycocyanin, tricolor, cyanine, etc., chemiluminescent substances (such as luminol), haptens (dinitrofluorobenzene, adenosine monophosphate (AMP), 2,4- Dinitroaniline, etc.), metal particles (ferritin particles, colloidal gold particles, etc.), specific binding pairs (biotin and avidins (eg, streptavidin), lectins and sugar chains, agonists and agonist receptors, HEP and antithrombin III ( ATIII )) And the like.

The antibody can be detected by detecting a signal of a labeling substance that is directly bound to the antibody. Signal detection can be performed by a person skilled in the art as appropriate from a known method according to the type and state of the sample used, the type of labeling substance, and the like. For example, when a cell itself is used as a sample, a dye, density of metal particles, radioactivity count, fluorescence intensity, fluorescence polarization, emission intensity, etc. may be detected using a microscope. In addition, when a cell suspension is used as a sample, a technique such as a microscope or flow cytometry can be employed. Further, when the extracted sugar chain is used as a sample, techniques such as immunoblotting and ELISA can be employed. See the Examples for a specific example.
From the standpoint of rapidity and simplicity, the sugar chain structure is preferably detected using an antibody.

  Whether or not the GlcN residue in the HEP skeleton or HS skeleton present in the cell has increased depends on whether apoptosis has been induced in the cells to be detected for apoptosis (normal: negative control) and apoptosis has occurred. It can be examined by comparing with what may have been. That is, the detection result of the sugar chain structure (the amount of the sugar chain structure) in a cell in which apoptosis may be induced is a cell in which apoptosis is not induced (normal) If it exceeds the detection result (the amount of the sugar chain structure) of the sugar chain structure in, the determination can be made and detected as an “increase” of the sugar chain structure in the former cell. As a result, the cells in which the increase in the sugar chain structure is detected can be determined and detected as those in which apoptosis is induced.

  The detection method 1 of the present invention may further include other steps as long as it includes at least the “step of detecting an increase in GlcN residues in the HS skeleton present in cells” as described above. For example, steps necessary for carrying out the present invention may be added as appropriate, such as a step of extracting a sugar chain from cells to be detected for apoptosis and a step of statistical analysis.

<2> Detection Agent of the Present Invention The detection agent of the present invention is an apoptosis detection agent comprising as an active ingredient an antibody that binds to a GlcN residue in the HEP skeleton or HS skeleton.
The description of “an antibody that binds to a GlcN residue in the HEP skeleton or HS skeleton”, which is an active ingredient of the detection agent of the present invention, is the same as in <1> above. That is, an antibody that can be used in the detection method 1 of the present invention can be used as an active ingredient of the detection agent of the present invention as it is. Needless to say, a labeling substance may be directly bound to the antibody.
The detection agent of the present invention may further contain other components as long as it contains “an antibody that binds to a GlcN residue in the HEP skeleton or HS skeleton” as an active ingredient. Examples of such other components include reagent-acceptable stabilizers, preservatives, excipients, and the like.
The detection agent of the present invention can be prepared as a normal reagent / preparation, etc. by adding “an antibody that binds to a GlcN residue in the HEP skeleton or HS skeleton” as it is or by adding other components as described above as necessary. It can be manufactured according to the method.
The detection agent of the present invention can be used according to the method described in the detection method 1 of the present invention in <1>.

<3> Kit of the present invention The kit of the present invention is an apoptosis detection kit containing at least the components shown in the following (1) and (2);
(1) an antibody that binds to a GlcN residue in the HEP skeleton or HS skeleton,
(2) A reagent for detecting the antibody of (1) above.
The description of (1) “an antibody that binds to a GlcN residue in the HEP skeleton or HS skeleton”, which is a component of the kit of the present invention, is the same as in <1> above. That is, an antibody that can be used in the detection method 1 of the present invention can be used as it is as an antibody related to the component (1) of the kit of the present invention. Needless to say, a labeling substance may be directly bound to the antibody.

The “reagent for detecting the antibody of (1)”, which is another component of the kit of the present invention, is a reagent that can detect “an antibody that binds to a GlcN residue in the HEP skeleton or HS skeleton” in some way. As long as there is no particular limitation. As such a reagent, for example, the secondary antibody described in the above <1> (“antibody that binds to a GlcN residue in the HEP skeleton or HS skeleton” (primary antibody) and a labeling substance are bound). An antibody (an antibody that binds to an immunoglobulin related to the primary antibody) can be exemplified. In the case where “an antibody that binds to a GlcN residue in the HEP skeleton or HS skeleton” is directly bound to a labeling substance, for example, when the labeling substance is an enzyme, “the antibody of (1) above” The “reagent for detecting” may be a substrate (such as a chromogenic substrate) of the enzyme.
The kit of the present invention can be used according to the detection method 1 of the present invention <1>.
As long as the kit of the present invention includes at least the components shown in the above (1) and (2), it may further contain other desired components. Examples of such other components include a cleaning solution and an enzyme reaction stop solution. Moreover, the positive control (QC control) for maintaining the implementation level between measurement batches to a fixed level can also be included.
These components can be stored in separate containers.

<4> Screening method 1 of the present invention
The screening method 1 of the present invention comprises an apoptosis-inducing agent comprising at least a step of contacting a test substance with a cell and selecting the test substance when the GlcN residue in the HEP skeleton or HS skeleton in the cell is increased or decreased. This is a screening method for candidate substances for apoptosis inhibitors.
The “test substance” as used herein means a substance to be subjected to screening for candidate substances for apoptosis inducers or apoptosis inhibitors.
In the screening method 1 of the present invention, first, this test substance is brought into contact with a target cell for apoptosis induction or apoptosis inhibition. The contact method is not particularly limited as long as the test substance molecule can come into contact with the cell. As an example of a simple method, for example, a method of adding a test substance to a culture solution containing cells can be exemplified.

When screening a candidate substance for an apoptosis inducer, the test substance and cells incubated in a medium capable of maintaining and growing the target cells may be brought into contact with each other and incubated.
In addition, when screening for a candidate substance for an apoptosis inhibitor, target cells are preliminarily placed in an environment in which apoptosis is induced (for example, in the presence of an apoptosis inducer such as daunomycin or in an environment in which serum components are deficient). In this case, the cells are brought into contact with the test substance, or the test substance is brought into contact with the target cell, and then incubated in an environment in which apoptosis is induced (see above).
Then, according to the detection method 1 of the present invention <1>, the GlcN residue in the HEP skeleton or HS skeleton in the cell may be detected.
In the screening of an apoptosis inducer candidate substance, if the sugar chain structure in the cell increases by contacting the test substance, apoptosis is induced. Therefore, the test substance is selected as the apoptosis inducer candidate substance. Can be selected.
Similarly, in screening for a candidate substance for an apoptosis inhibitor, if the sugar chain structure in the cell is reduced by bringing the test substance into contact with the test substance, apoptosis is inhibited. Can be selected as a candidate substance.

  The screening method 1 of the present invention comprises the above-mentioned “step of selecting the test substance when the test substance is brought into contact with the cell and the GlcN residue in the HEP skeleton or HS skeleton in the cell is increased or decreased”. Other steps may be further included as long as they are included. For example, the method may further include a step of subjecting the substance selected in this step (candidate substance for apoptosis inducer or apoptosis inhibitor) to other screening or testing.

<5> Detection method 2 of the present invention
The detection method 2 of the present invention is an apoptosis detection method including at least a step of detecting an increase in expression of at least one of NDST-3 and NDST-4 in a cell.
Expression of NDST-3 and NDST-4 in the cell may be detected as a protein or may be detected as mRNA. For detection as a protein, for example, an antibody or the like can be used. Moreover, when detecting as mRNA, RT-PCR using the specific primer about NDST-3 and NDST-4 which was illustrated in the Example, for example can also be employ | adopted. In particular, it is preferable to detect the expression of mRNA. Specific primers for NDST-3 and NDST-4 in this case can be designed based on the respective base sequences (NDST-3: SEQ ID NO: 19, NDST-4: SEQ ID NO: 21). Specific examples include a combination of SEQ ID NOs: 5 and 6 for NDST-3 and a combination of SEQ ID NOs: 7 and 8 for NDST-4. Moreover, what was described in the Example can be illustrated also about specific examples, such as conditions of RT-PCR.

  Whether or not the expression of NDST-3 or NDST-4 in the cell is increased depends on whether apoptosis is induced (normal: negative control) and apoptosis is induced in the cell to be detected for apoptosis. It can be examined by comparing with what may be present. That is, the expression level of NDST-3 or NDST-4 in a cell in which apoptosis may be induced is NDST-3 or in a cell in which apoptosis is not induced (normal). If it exceeds the expression level of NDST-4, it can be determined and detected as an “increase” in expression. Then, the cells in which the increase in expression is detected can be determined and detected as those in which apoptosis is induced.

<6> Screening method 2 of the present invention
The screening method 2 of the present invention includes at least a step of selecting the test substance when the test substance and the cell are brought into contact with each other and thereby the expression of at least one of NDST-3 and NDST-4 in the cell is increased or decreased. A screening method for a candidate substance for an apoptosis inducer or apoptosis inhibitor. When the expression of at least one of NDST-3 and NDST-4 increases, the test substance can be selected as an apoptosis inducer, and when the expression of at least one of NDST-3 and NDST-4 decreases The test substance can be selected as an apoptosis inhibitor.
In the screening method 2 of the present invention, instead of “increase or decrease in GlcN residues in the HEP skeleton or HS skeleton” which is the screening index of the screening method 1 of the present invention, the expression of at least one of NDST-3 and NDST-4 is "Increase or decrease" is adopted as a screening index. Therefore, the description other than this point is the same as the screening method 1 of the present invention.
Then, the expression of at least one of NDST-3 and NDST-4 may be detected according to the detection method 2 of the present invention <5>. Therefore, “expression” in the screening method 2 of the present invention is also preferably the expression of mRNA, and other explanations relating to this are the same as in <5> above.

Hereinafter, the present invention will be described in detail by way of examples.
EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, this does not limit the technical scope of this invention.

1. 1. Materials and methods 1-1. Cells used in this example The following various cell lines were used as samples derived from living tissue.
(1) K562 strain: A cell line derived from human chronic myeloid leukemia (Blood, 1975 Mar; 45 (3): 321-334) (RCB0027).
(2) HL-60 strain: Human promyelocytic leukemia cell line (Nature, 1977 Nov 24; 270 (5635):
347-349) (RCB0041).
(3) U937 strain: a human lymphoma cell line (J. Exp. Med., 1976 Jun 1; 143 (6): 1528-1533) (RCB1978).
(4) C-1 strain: A human colon cancer-derived cell line (Cancer Res. 2001 Jun 1; 61 (11): 4620-4627).
(5) LS174T strain: A human colon cancer-derived cell line (Clin. Cancer Res., 2006 Mar 1; 12 (5): 1606-1614).
(6) PC12 strain: A cell strain derived from rat pheochromocytoma and has the property of extending and differentiating neurites by adding NGF (Natl. Acad. Sci. USA., 1976 Jul; 73 (7): 2424-2428) (RCB0009).
(7) Jurkat strain: a human T cell line (Int. J. Cancer, 1977 May 15; 19 (5): 621-626) (RCB0806).
The numbers starting with “RCB” are cell numbers in RIKEN CELL BANK, RIKEN BioResource Center.
For the C-1 strain, LS174T strain and PC12 strain, DMEM containing 10% FCS and RPMI-1640 medium containing 10% FCS were used as cell culture solutions for the other cell lines.

1-2. Antibody (1) JM403 antibody used in this example: The one manufactured by Seikagaku Biobusiness Co., Ltd. was used. The immunoglobulin class of this antibody is mouse IgM, which binds to a GlcN residue present in the HEP or HS backbone. When the antigen is treated with heparitinase, the binding property is lost, and it does not substantially bind to HA, CS, DS, and KS.

(2) NAH43 antibody: The National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (currently the National Institute for Product Evaluation Technology Patent Biological Depositary) has accession number FERM BP
The product produced by the hybridoma commissioned as -10535 was used. The immunoglobulin class of this antibody is mouse IgM. This antibody binds to a GlcN residue present in the HEP skeleton or the HS skeleton, but includes EHS-HS, HA, Ch, various CS (CS-A, CS-B, CS-C, CS-D, CS-E). ) And KS.

(3) NAH46 antibody: manufactured by Seikagaku Biobusiness Corporation. The immunoglobulin class of this antibody is mouse IgM, which binds to GlcNAc present in the HEP or HS backbone. When the antigen is treated with heparitinase, the binding property is lost and it does not bind to HA, CS, DS, or KS. (It can also be produced by the method described in WO2006 / 106950, and on March 11, 2005, the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (currently the National Institute for Product Evaluation Technology Patent Biology) It can also be produced from a hybridoma deposited at the Deposit Center) under the deposit number FERM BP-10536).

(4) 10E4 antibody: The one manufactured by Seikagaku Biobusiness Co., Ltd. (trade name: F58-10E4) was used. The immunoglobulin class of this antibody is mouse IgM, which binds to a GlcNS residue present in HS. When the antigen is treated with heparitinase, the binding property is lost, and it does not substantially bind to HA, CS, DS, KS, and DNA.

(5) HepSS-1 antibody: The one manufactured by Seikagaku Biobusiness Co., Ltd. was used. The immunoglobulin class of this antibody is mouse IgM, which binds to O-sulfated / N-acetylated glucosamine present in HS. It also binds to various normal cells derived from mice, monkeys, rats, hamsters and chickens. HA, Ch, HEP, chondroitin-4-sulfate, chondroitin-6-sulfate, DS, CS-D, CS-E and KS are not substantially bound.

(6) ACH55 antibody: On March 1, 2006, the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (currently, the National Institute of Technology and Evaluation, Patent Biological Depositary Center) has accession number FERM BP-1079 What was produced by the commissioned hybridoma was used. The immunoglobulin class of this antibody is mouse IgM. Although this antibody binds to ACH, AS, HEP, HS, NAH and EHS-HS, HA, various CS (CS-A (W), CS-A (S), CS-B, CS-C, CS- D, CS-E) and KS are not substantially bound. Moreover, this antibody reacts strongly with CDSH among HEP derivatives and reacts very weakly with 2SH, 6SH and Ac-NSH, but NH ₂ -HEP, N
Ac-HEP, 6DSH, Ac- 6DSH, NH 2 -6SH, NH 2 -2SH, NSH, but does not substantially bind to NH ₂ -CDSH.
Since ACH which strongly reacted with the ACH55 antibody has an iduronic acid unit (-[IdoA-GlcNAc]-) as a main constituent disaccharide, it is considered that the epitope of the ACH55 antibody is composed of an iduronic acid unit. This is supported by the fact that this antibody also reacts strongly with CDSH (polysaccharide mainly composed of glucuronic acid units and iduronic acid units, and the abundance ratio of iduronic acid units is as high as 60% or more).
The NAH to which this antibody did not bind is the glucuronic acid unit (-[GlcA-GlcNAc]-)
The uronic acid C5-epimer of ACH consisting of Iduronic acid residue is particularly essential for antigen recognition of this antibody. Furthermore, since this antibody does not show binding to AS (the 2-position hydroxyl group of iduronic acid residue is sulfated), sulfation of iduronic acid (IdoA (2S)) It is suggested to inhibit.
The binding of this antibody is also due to N-deacetylation of the GlcNAc residue (NH ₂ -CDSH).
It was also suggested that the amino group of the GlcN residue was acetylated because it was lost by N-sulfation (NSH) and was also important in antigen recognition of this antibody. The reason why this antibody that does not bind to AS reacted weakly with 2SH, 6SH or Ac-NSH is that HEP undergoes multiple modifications such as N-desulfation, 2-O desulfation, and 6-O desulfation, It is presumed that the iduronic acid unit was manifested in the HEP derivative.

(7) AS22 antibody: As of March 1, 2006, the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (currently, the National Institute of Technology and Evaluation, Patent Biological Depositary Center) has accession number FERM BP-10775. What was produced by the commissioned hybridoma was used. The immunoglobulin class of this antibody is mouse IgM.
This antibody reacts strongly to AS but does not substantially bind to HS and HEP. This antibody can be any one of ACH, EHS-HS, KS, HA, NAH, CS-A (W), CS-A (S), CS-B, CS-C, CS-D, CS-E and Ch. Does not substantially bind to. Therefore, it is suggested that the epitope of this antibody contains IdoA (2S) residue.
In addition, in the HS and HEP molecules to which this antibody does not exhibit binding, α (1-4) bond of IdoA (2S) is present, but most of it is bound to GlcNS or O-sulfated GlcNS. Since it exists as a disaccharide unit, it is presumed that modification of the GlcN residue is also a factor that inhibits binding of this antibody.
In addition, this antibody does not substantially bind to NH ₂ -HEP and 6DSH, but shows the same degree of binding to NAc-HEP and Ac-6DSH obtained by N-acetylating them. This suggests that the epitope of this antibody contains a GlcN residue modified in some way at the N-position, particularly an N-acetylated GlcN residue. In addition, the binding of this antibody to NAc-HEP and Ac-6DSH suggests that O-sulfation or N-sulfation of the GlcN residue does not substantially affect the binding properties of this antibody. .

(8) LY111 antibody: The one manufactured by Seikagaku Biobusiness Co., Ltd. was used. The immunoglobulin class of this antibody is mouse IgM, which binds to chondroitin-4-sulfate. It does not substantially bind to HA, Ch, HEP, HS, DS and KS.

(9) G152 antibody (binding to 6-sulfated sialyl Lewis X ceramide, but 6'-sulfated sialyl Lewis X ceramide, 6,6'-bis-sulfated sialyl Lewis X ceramide, 6-sulfated Lewis X
Antibodies that do not bind to ceramide, Lewis X ceramide, etc. It was produced by the method described in JP-A-11-313684. )

1-3. Apoptosis induction treatment The induction of apoptosis for the various cells described above was performed by any of the following methods.
(1) Treatment with daunomycin (also referred to as daunomycin or daunorubicin) Apoptosis was induced by adding daunorubicin hydrochloride (manufactured by Sigma) to the cell culture medium.
(2) Serum deficiency treatment Apoptosis was induced by culturing the cells in a cell culture medium with an FCS content (usually 10%) of 0.6%.

1-4. Confirmation of Apoptosis Induction of apoptosis was confirmed by observing the ladder (fragmentation) of the chromosomal DNA of the cells by agarose gel electrophoresis.

1-5. Flow cytometry The above-mentioned various antibodies (primary antibodies) are added to a suspension of each of the above cells (one that does not induce apoptosis and one that induces apoptosis) to a final concentration of 5 μg / ml or 20 μg / ml. And incubated at 4 ° C. for 30 minutes. Thereafter, the resultant was washed with PBS containing FCS at a final concentration of 2%, and then a secondary antibody (FITC-labeled anti-mouse immunoglobulin antibody (Chemicon; catalog number AQ326F)) diluted to 1/200 was added and added at 30 ° C. at 30 ° C. Incubated for minutes. Thereafter, the plate was washed with PBS containing FCS having a final concentration of 2%, and analyzed by flow cytometry. In addition, what was incubated only with the secondary antibody without using the primary antibody was used as a control.

1-6. Expression analysis of NDST mRNA 1-3. Expression of mRNA of various NDSTs (NDST-1, NDST-2, NDST-3, NDST-4) in the K562 strain treated with daunomycin (final concentration 1.0 μM) by the method described in (1) Analyzed by PCR. The detailed conditions are as follows.
Each of the above cells was cultured in the presence or absence of daunomycin using a cell culture dish having a diameter of 100 mm. Thereafter, the culture supernatant was removed, and the cells were washed with PBS (−). 1 ml of ISOGEN (Nippon Gene Co., Ltd.) was added to the washed cells to lyse the cells. The cell lysate was extracted with chloroform, the RNA fraction was separated, and total RNA was separated by isopropanol precipitation. The recovered RNA was dissolved in DEPC-treated water (RNase-free water; Nacalai Tesque, Inc.) to obtain an RNA solution.
The synthesis of cDNA was performed using SuperScript II Reverse Transcription kit (Invitrogen). 5 μg total RNA, 1 mM dNTP mixture, 500 ng oligo (dT) 12-18 primer and DEPC-treated water were mixed in a total volume of 10 μl reaction solution, heated at 65 ° C. for 5 minutes, and then rapidly cooled on ice. This reaction solution was mixed with 1 × reaction buffer, 5 mM MgCl ₂ , 10 mM DTT and 40 U RNaseOUT (registered trademark) attached to the kit to make a total volume of 20 μl. After heating at 42 ° C. for 2 minutes, 200 U of SuperScript II Reverse Transcriptase (reverse transcriptase; Invitrogen) was added, and the mixture was further incubated for 50 minutes. Thereafter, the reverse transcriptase was inactivated by heating at 70 ° C. for 15 minutes. Thereafter, RNase H was added and incubated at 37 ° C. for 20 minutes to decompose the total RNA used as a template for cDNA synthesis. The cDNA thus obtained was used for the next experiment.
A partial sequence of the target gene was amplified using the cDNA obtained above and each gene-specific primer shown below. In addition, the used primer is as follows. “F:” indicates a forward primer, and “R:” indicates a reverse primer. The significance of the temperature described in parentheses will be described later.

(1) NDST-1 (59 ° C., 397 bp)
F: 5'-ACCTGTCCAACTATGGGAATGACC-3 '(SEQ ID NO: 1)
R: 5'-AACTCCATGTACCAGTCGATGCCT-3 '(SEQ ID NO: 2)

(2) NDST-2 (58 ° C., 307 bp)
F: 5'-TCTCGTGAACTAGACCGGAGCAT-3 '(SEQ ID NO: 3)
R: 5'-GACGATCACAGGTTTTCTCCTTGGAC-3 '(SEQ ID NO: 4)

(3) NDST-3 (58 ° C., 310 bp)
F: 5'-ACTATGGGAATGACCGACTGGGAT-3 '(SEQ ID NO: 5)
R: 5'-GGGGAGTTACTAAGGATGGAAGGATG-3 '(SEQ ID NO: 6)

(4) NDST-4 (59 ° C., 397 bp)
F: 5'-TGAGCAGAAAGACCCTCTATGGCA-3 '(SEQ ID NO: 7)
R: 5'-TGAGGGGTCAATGAGGATGGTGAT-3 '(SEQ ID NO: 8)

(5) HS2ST (60 ° C., 444 bp)
F: 5'-CAGGATTTTATCATGGACACG-3 '(SEQ ID NO: 9)
R: 5'-TCTTTCCTGTGCGATAGAGT-3 '(SEQ ID NO: 10)

(6) HS6ST-1 (60 ° C., 490 bp)
F: 5'-TGGACCGAGCTCACCAACTG-3 '(SEQ ID NO: 11)
R: 5'-AGGGCCGGATGAACTTGAGG-3 '(SEQ ID NO: 12)

(7) HS6ST-2 (60 ° C., 333 bp)
F: 5'-TGGACCGAGCTCACCAACTG-3 '(SEQ ID NO: 13)
R: 5'-AGGGCCGGATGAACTTGAGG-3 '(SEQ ID NO: 14)

(8) HS6ST-3 (60 ° C., 398 bp)
F: 5'-TAGCTGCAAAGCGGGTCAGAAG-3 '(SEQ ID NO: 15)
R: 5'-TGTTAGCCAGGTTGTAGGTGCAATCC-3 '(SEQ ID NO: 16)

(9) G3PDH (57 ° C., 498 bp)
F: 5'-AAGGTCATCCATGACAAC-3 '(SEQ ID NO: 17)
R: 5'-CACCCTGTTGCTGTAGCCA-3 '(SEQ ID NO: 18)

  The PCR reaction was performed using 1 × AmpliTaq reaction buffer, 1 mM dNTP mixture, 0.5 μM forward primer, 0.5 μM reverse primer, 250 ng cDNA (RNA equivalent), 2.5 U AmpliTaq Gold (registered trademark) DNA polymerase (Applied Biosystems ( Applied Biosystems)) and 50 μl of a reaction solution containing autoclave water, and using GeneAmp9700 (Applied Biosystems). Amplification was performed at a temperature of 94 ° C. (30 seconds), X ° C. (temperature indicated in the above primer pair) (30 seconds), 72 ° C. (30 seconds) for the genes (1) to (4). Was repeated 35 times and (5) to (8) were repeated 30 times. However, G3PDH was repeated 25 times. The PCR product was electrophoresed using a 2% agarose gel, and the amplified DNA fragment was detected.

2. Result 2-1. Confirmation of Apoptosis Induction of apoptosis of all cells treated by the method of “1-3.” Was observed by agarose gel electrophoresis, and it was confirmed that apoptosis was induced. As an example, FIG. 1 shows an electrophoresis image when the K562 strain was treated with daunomycin at a final concentration of 1 μM for 2 days. In FIG. 1, “M” lanes indicate molecular weight markers, “−” lanes indicate cells not treated with daunomycin, and “+” indicate cells treated with daunomycin.
In “+” of FIG. 1, it was shown that the chromosomal DNA of the cell was fragmented and apoptosis was induced.

2-2. The flow cytometry results are shown in FIGS. In each figure, the horizontal axis of the graph indicates the fluorescence intensity, and the vertical axis indicates the number of cells. Moreover, in each figure, the mountain-shaped solid line which exists in the left position in a graph shows control. When the solid line peak is shifted to the right side of this control, the primary antibody is bound to the cell (the cell is stained with the primary antibody).

The explanation of each figure is as follows. In addition, the antibody shown below is an antibody used as a primary antibody. The concentration in parentheses is the final concentration of the antibody when the primary antibody is reacted. Fig. 2: Results using the K562 strain. A to E are cells not treated with daunomycin, F to J are cells treated with daunomycin at a final concentration of 2 μM for 2 days, and K to O are cells treated with daunomycin at a final concentration of 5 μM for 2 days. .
A: JM403 antibody (5 μg / ml)
B: NAH46 antibody (50 μg / ml)
C: NAH43 antibody (20 μg / ml)
D: NAH43 antibody (50 μg / ml)
E: NAH43 antibody (100 μg / ml)

F: JM403 antibody (5 μg / ml)
G: NAH46 antibody (50 μg / ml)
H: NAH43 antibody (20 μg / ml)
I: NAH43 antibody (50 μg / ml)
J: NAH43 antibody (100 μg / ml)

K: JM403 antibody (5 μg / ml)
L: NAH46 antibody (50 μg / ml)
M: NAH43 antibody (20 μg / ml)
N: NAH43 antibody (50 μg / ml)
O: NAH43 antibody (100 μg / ml)

Fig. 3: Results using HL60 strain. A to C show cells not treated with daunomycin, and D to F show cells treated with daunomycin at a final concentration of 3 μM for 2 days.
A: 10E4 antibody (5 μg / ml)
B: NAH46 antibody (5 μg / ml)
C: JM403 antibody (5 μg / ml)

D: 10E4 antibody (5 μg / ml)
E: NAH46 antibody (5 μg / ml)
F: JM403 antibody (5 μg / ml)

Fig. 4: Results using U937 strain. A to C are cells cultured for 4 days in the presence of 10% FCS, and D to F are cells treated for 4 days under serum-deficient conditions (in the presence of 0.6% FCS).
A: 10E4 antibody (5 μg / ml)
B: NAH46 antibody (5 μg / ml)
C: JM403 antibody (5 μg / ml)

D: 10E4 antibody (5 μg / ml)
E: NAH46 antibody (5 μg / ml)
F: JM403 antibody (5 μg / ml)

Fig. 5: Results using U937 strain. A to C are cells not treated with daunomycin, and D to F are cells treated with daunomycin at a final concentration of 0.2 μM for 2 days.
A: 10E4 antibody (5 μg / ml)
B: NAH46 antibody (5 μg / ml)
C: JM403 antibody (5 μg / ml)

D: 10E4 antibody (5 μg / ml)
E: NAH46 antibody (5 μg / ml)
F: JM403 antibody (5 μg / ml)

FIG. 6 shows the results using the C-1 strain. In either case, the cells were cultured for 4 days in the presence of 10% FCS.
A: AS22 antibody (5 μg / ml)
B: ACH55 antibody (5 μg / ml)
C: 10E4 antibody (1 μg / ml)
D: NAH46 antibody (1 μg / ml)
E: JM403 antibody (1 μg / ml)
F: HepSS-1 antibody (1 μg / ml)

FIG. 7 shows the results using the C-1 strain. In either case, the cells were cultured for 4 days under serum-deficient conditions (in the presence of 0.6% FCS).
A: AS22 antibody (5 μg / ml)
B: ACH55 antibody (5 μg / ml)
C: 10E4 antibody (1 μg / ml)
D: NAH46 antibody (1 μg / ml)
E: JM403 antibody (1 μg / ml)
F: HepSS-1 antibody (1 μg / ml)

FIG. 8 shows the results using the LS174T strain. In either case, the cells were cultured for 4 days in the presence of 10% FCS.
A: AS22 antibody (5 μg / ml)
B: ACH55 antibody (5 μg / ml)
C: 10E4 antibody (1 μg / ml)
D: JM403 antibody (1 μg / ml)
E: NAH46 antibody (1 μg / ml)
F: HepSS-1 antibody (5 μg / ml)
G: LY111 antibody (5 μg / ml)
H: G152 antibody (20-fold dilution)

FIG. 9 shows the results using the LS174T strain. In either case, the cells were cultured for 4 days under serum-deficient conditions (in the presence of 0.6% FCS).
A: AS22 antibody (5 μg / ml)
B: ACH55 antibody (5 μg / ml)
C: 10E4 antibody (1 μg / ml)
D: JM403 antibody (1 μg / ml)
E: NAH46 antibody (1 μg / ml)
F: HepSS-1 antibody (5 μg / ml)
G: LY111 antibody (5 μg / ml)
H: G152 antibody (20-fold dilution)

FIG. 10 shows the results using the PC12 strain. A to C are cells cultured for 5 days in the presence of 10% FCS, and D to F are cells treated for 5 days under serum-deficient conditions (in the presence of 0.6% FCS).
A: 10E4 antibody (5 μg / ml)
B: NAH46 antibody (5 μg / ml)
C: JM403 antibody (5 μg / ml)

D: 10E4 antibody (5 μg / ml)
E: NAH46 antibody (5 μg / ml)
F: JM403 antibody (5 μg / ml)

FIG. 11 shows the results using PC12 strain. A to C are cells not treated with daunomycin, and D to F are cells treated with daunomycin at a final concentration of 1 μM for 2 days.
A: 10E4 antibody (5 μg / ml)
B: NAH46 antibody (5 μg / ml)
C: JM403 antibody (5 μg / ml)

D: 10E4 antibody (5 μg / ml)
E: NAH46 antibody (5 μg / ml)
F: JM403 antibody (5 μg / ml)

Fig. 12: Results using the Jurkat strain. A to C are cells cultured for 5 days in the presence of 10% FCS, and D to F are cells treated for 4 days under serum-deficient conditions (in the presence of 0.6% FCS).
A: 10E4 antibody (5 μg / ml)
B: NAH46 antibody (5 μg / ml)
C: JM403 antibody (5 μg / ml)

D: 10E4 antibody (5 μg / ml)
E: NAH46 antibody (5 μg / ml)
F: JM403 antibody (5 μg / ml)

Figure 13: Results using Jurkat strain. A to C are cells not treated with daunomycin, and D to F are cells treated with daunomycin at a final concentration of 1 μM for 2 days. A: 10E4 antibody (5 μg / ml)
B: NAH46 antibody (5 μg / ml)
C: JM403 antibody (5 μg / ml)

D: 10E4 antibody (5 μg / ml)
E: NAH46 antibody (5 μg / ml)
F: JM403 antibody (5 μg / ml)

From FIG. 2 to FIG. 13, the staining characteristics varied depending on the type of cell line, the presence or absence of induction of apoptosis, and the type of primary antibody used. However, when the JM403 antibody was used as a primary antibody, it was shown that the staining in cells in which apoptosis was induced was increased in common with all cell lines compared to cells in which apoptosis was not induced. The same was true when NAH43 antibody was used.
From these facts, in the cells in which apoptosis is induced, the expression of the antigen (GlcN residue in the HEP skeleton or HS skeleton) against the JM403 antibody or NAH43 antibody may increase as compared with the cells in which apoptosis is not induced. Indicated.

2-3. Expression analysis of NDST mRNA The results of various NDST mRNA expression analyzes by RT-PCR are shown in FIG. In FIG. 14, lane “C” is the result of K562 strain not treated with daunomycin, lane “1” is the result of K562 strain treated with daunomycin for 1 day, and lane “2” is K562 treated with daunomycin for 2 days. The results for each strain are shown. G3PDH is a RT-PCR control.
From FIG. 14, NDST-1 and NDST-2 did not change their expression levels regardless of the presence or absence of apoptosis induction treatment (daunomycin treatment). On the other hand, for NDST-3 and NDST-4, it was shown that their expression was induced by apoptosis-inducing treatment (daunomycin treatment).
Although not shown in the drawings, no mRNA expression was observed for HS6ST-2 and HS6ST-3 regardless of the presence or absence of apoptosis induction treatment (daunomycin treatment). As for HS6ST-1, the expression level of mRNA was not changed on the first day by the apoptosis induction treatment (daunomycin treatment), but the expression level was decreased on the second day. As for HS2ST, the expression level of mRNA was not changed on the first day by the apoptosis induction treatment (daunomycin treatment), but the expression level was unchanged or slightly decreased on the second day.

3. Production of the kit of the present invention A kit of the present invention comprising the following components was produced.
(1) One JM403 antibody (primary antibody)
(2) One FITC-labeled anti-mouse immunoglobulin antibody (secondary antibody)
(3) One cleaning solution (PBS)

Moreover, this invention kit which consists of the following structural components was manufactured.
(1) One NAH43 antibody (primary antibody)
(2) One FITC-labeled anti-mouse immunoglobulin antibody (secondary antibody)
(3) One cleaning solution (PBS)

Claims (4)

  A method for detecting apoptosis, comprising at least a step of detecting an increase in expression of at least one of NDST-3 and NDST-4 in a cell.   The detection method according to claim 1, wherein the expression is mRNA expression.   An apoptosis-inducing agent or apoptosis-inhibiting agent comprising at least a step of contacting a test substance with a cell and selecting the test substance when the expression of at least one of NDST-3 and NDST-4 in the cell increases or decreases Candidate substance screening method.   The screening method according to claim 3, wherein the expression is mRNA expression.

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