JPWO2005082933A1 - Novel chimeric protein, gene encoding the same, and leukemia discrimination means using these genes and proteins - Google Patents

Abstract

The present invention clarifies the genetic essence in different types of acute lymphocytic leukemia (ALL), provides a detection means that can distinguish the type of ALL at the gene level before full-scale treatment, The invention aims to provide ALL patients with an opportunity to be treated as appropriately as possible. The present inventor has determined that a novel chimeric protein obtained by binding a part of MEF2D protein and a part of DAZAP1 protein is specific to a specific type of precursor B cell ALL, A chimeric gene and a chimeric protein, which provide a novel chimeric gene encoding the same, and further determine the precursor B cell ALL of the sample provider by detecting the chimeric gene or the chimeric protein in the sample It has been found that the above object can be achieved by providing a detection method. The present invention also provides a screening method for a therapeutic agent for leukemia using the above chimeric gene or chimeric protein.

Description

  The present invention relates to a novel chimeric protein that enables detailed discrimination of precursor B-cell acute lymphoblastic leukemia (also referred to as ALL), a gene encoding the chimeric protein, and the chimeric protein or gene The present invention relates to a detection method that makes it possible to determine the ALL to be used.

Acute leukemia is classified into acute myelogenous leukemia (AML), acute mixed leukemia, and acute lymphoblastic leukemia (ALL). ALL is a precursor (or lymphoblastic) malignant tumor. In progenitor B-cell ALL, genetic abnormalities associated with chromosomal translocation are the cause, and so far t (1; 22); BCL / ABL, t (v; 11q23); MLL, t (1; 19); E2A / Translocations such as PBX1 and t (12; 21); TEL / AML1 are known.
The most common childhood leukemia is ALL resulting from genetic abnormalities of progenitor B cells, which has a relatively good prognosis for chemotherapy (Greaves, MF & Wiemels, J .: Origins of chromosome translocations in (childhood leukaemia, Nat. Rev. Cancer, 3 , 639-649, 2003). The main subtype of ALL is caused by gene abnormalities due to single base mutations and deletions, but it is often caused by changes in the entire chromosome associated with hyperdiposy and chromosomal translocation. In chromosomal translocation, genes on different chromosomes are located close to each other, and both chimeric (or fusion) genes are often rearranged. As a result, fusion mRNA is expressed under transcription control different from the conventional one. Are translated into hybrid proteins with different roles. About 200 genes or more are known to be involved in translocation of childhood leukemia. Among them, certain genes are strongly involved in canceration, but many other genes are rare. Only involved in canceration.
The most common common translocation of childhood progenitor B cells ALL is t (1; 19) (q23; p13), which usually causes fusion between the PBX1 gene on chromosome 1q23 and the E2A gene on chromosome 19p13.3 . The wild type E2A gene product is essential for B cell maturation. The chimeric gene E2A-PBX1 encodes a fusion protein of the transcriptional activation motif of E2A and the DNA-binding homeodomain of PBX1. Although the same E2A-PBX1 fusion gene is detected in 95% or more cases of ALL, in rare cases, the fusion gene may not be detected (Hunger, SP, Galili, N.,). Carroll, AJ, Crist, WM, Link, MP & Clear, ML: Thet (1; 19) (q23; p13) results in consistent fusion of E2A and PBX1 coding sequences acute lymphoblastic leukemias, Blood, 77, 687-693,1991; Izraeli, S., Janssen, J.W.G., Haas, O.A., Harbott, J., Brok-Simoni, F., Walther J. U., Kovar, H., Henn, T., Ludwig, WD, Reiter, A., Rechavi, G., Bartram, CR, Gadner, H. & Lion, T .: Detection and clinical relevance of genetic abnormalities in pediatric acute lymphoblastic leukemia: a comparison between cytogenetic and polymerase chain reaction analyses.Leukemia, 7, 671-678,1993). In fact, E2A gene is not involved in translocation in early progenitor B cell ALL, CD-34 positive B cell progenitor ALL, normal ALL, T-ALL, B cell lymphoma / leukemia, acute myeloid leukemia (AML) Examples have been reported and this fact indicates that in these patients the chromosomal t (1; 19) translocation is not due to rearrangement of the E1A and PBX genes.

In this way, it is becoming clear that there are various types of ALL at the gene level even if it is called ALL. In spite of this, in general, it is imperative to treat patients with different types of ALL with a low response rate if only treatments with a good response rate are performed uniformly. It is not appropriate.
Therefore, the problem to be solved by the present invention is to clarify the genetic nature of such different types of ALL, and to provide a detection means that can discriminate the type of ALL at the gene level before full-scale treatment. But it is to give many ALL patients the opportunity to receive the most appropriate treatment possible.
In order to respond to this problem, the present inventor has a progenitor B cell acute leukemia cell line (however, a chromosome t (1; 19) translocation established from the peripheral blood of pediatric ALL patients using conventional methods. However, the gene level of the E2A-PBX fusion gene was not generated (TS-2 cells) was examined, and the location where translocation occurred in this cell line was accurately identified. That is, the present inventor elucidated that fusion of the MEF2D gene of chromosome 1q22 and the DAZAP1 gene of chromosome 19p13.3 occurred in TS-2 cells. Furthermore, mRNA of this chimeric gene was also detected in bone marrow cells of pediatric ALL patients from which the cells were isolated, as in the case of TS-2 cells.
Therefore, the present inventor has clarified that details of the cause of ALL can be determined at the molecular level using the present chimeric gene or a chimeric protein produced based thereon as an index. The present invention based on the above has been completed.
That is, the present invention provides a novel chimeric protein (hereinafter also referred to as the present chimeric protein) comprising a part of MEF2D protein and a part of DAZAP1 protein, and a novel chimeric gene encoding the chimeric protein. (Hereinafter also referred to as the present chimeric gene).
Furthermore, the present invention provides a method for detecting a chimeric gene and a chimeric protein (hereinafter referred to as “the chimeric gene” or “chimeric protein” in the sample), wherein the precursor B cell acute lymphoblastic leukemia of the sample provider is detected by detecting the present chimeric gene or the present chimeric protein. , Also referred to as this detection method).
1. This chimeric gene and chimeric protein
As described above, this chimeric gene is a chimeric gene obtained by fusing part of the MEF2D gene and part of the DAZAP1 gene. The MEF2D gene and the DAZAP1 gene are known human genes, respectively, and their base sequences are also registered (MEF2D gene: NM_005920 (SEQ ID NO: 1), DAZAP1 gene: NM_170711 (SEQ ID NO: 2)). However, the present chimeric gene, which is a fusion gene of these genes, is not known including the function of the present chimeric protein synthesized based on the gene.
As will be described later, it has been found that the present chimeric protein is localized in the cell nucleus and forms a heterodimer with a MEF2 family protein (such as HDAC4 protein). This chimeric protein binds to HDAC4 protein more strongly than MEF2D protein. It is presumed that this chimeric protein promotes the transcriptional activation of the target gene through this action and induces canceration.
The specific sequence of this chimeric gene is as follows: 1) the present chimeric gene having the nucleotide sequence shown in SEQ ID NO: 3 (MEF2D-DAZAP1 fusion gene), the corresponding chimeric protein (amino acid sequence shown in SEQ ID NO: 5), and 2) The present chimeric gene (DAZAP1-MEF2D fusion gene) having the nucleotide sequence shown in SEQ ID NO: 4 and the corresponding chimeric protein (amino acid sequence shown in SEQ ID NO: 6) can be exemplified.
The chimeric protein can be obtained by isolation and purification from cancerous cells of the subject ALL patient, but it is extremely preferable to obtain it as a recombinant when a large amount is required.
As described above, the present chimeric gene, which is a group for producing such recombinant chimeric protein, is prepared by a conventional method using, for example, PCR (Polymerase chain reaction) using the base sequence of the chimeric gene found by the present invention. The desired chimeric gene can be obtained as cDNA using RT (Reverse Transscriptase) -PCR method by extracting mRNA of cancerous cells of the subject ALL patient using a gene amplification method such as the above method .
In addition, the leukemia cell of the subject ALL patient can be easily obtained using a conventional method. Specifically, it can be obtained from, for example, bone marrow and peripheral blood. By routine phenotypic analysis, precursor B cell acute lymphoblastic leukemia cells can be identified using FAB classification. The B cell lineage can be determined by monitoring the expression of the surface marker CD19. For details, see Yoshinari, M .; , Imaizumi, M .; , Eguchi, M .; Ogasawara, M .; Saito, T .; Suzuki, H .; , Koizumi, Y .; , Cui, Y .; , Sato,. Saisho, T .; , Ichinohasama, R .; , Matsubara, Y .; , Kamada, N .; & Iinuma, K .; : Establishment of a novel cell line (TS-2) of pre-B accurate lymphoma with at (1; 19) not involving the E2A gene. , Cancer Genet. Cytogenet. 101, 95-102, 1998.
Furthermore, the phosphite-triester method (Ikehara, M., et al., Proc. Natl. Acad. Sci. USA, 81 , 5956 (1984)) can be used to chemically synthesize this chimeric gene, and this chimeric gene can be synthesized using a DNA synthesizer to which these chemical synthesis methods are applied. You can also. In this case, the chimeric gene can be obtained by synthesizing a plurality of DNAs having a part of the chimeric gene, ligating them together, inserting the DNA into an appropriate vector, and transforming Escherichia coli. .
By using the present chimeric gene obtained as described above, a recombinant present chimeric protein can be produced according to a general gene recombination technique.
More specifically, the chimeric gene is incorporated into a gene expression vector in an expressible form of the chimeric gene, the recombinant vector is introduced into a host corresponding to the properties of the gene expression vector, The desired chimeric protein can be produced by transforming and culturing such a transformant.
In general, the gene expression vector used here preferably has a promoter, an enhancer, etc. in the upstream region of the gene to be expressed and a transcription termination sequence in the downstream region.
The expression of the chimeric gene is not limited to a direct expression system, and for example, a fusion protein expression system using a hexahistidine gene, a glutathione-S-transferase gene, a maltose binding protein gene, or a thioredoxin gene can be used. is there.
Examples of the vector for gene expression include pQE, pGEX, pT7-7, pMAL, pTrxFus, pET, pNT26CII, etc., assuming that the host should be Escherichia coli. Moreover, as what should make a host Bacillus subtilis, pPL608, pNC3, pSM23, pKH80 etc. can be illustrated.
Moreover, as what should make a host into a fermenter, pGT5, pDB248X, pART1, pREP1, YEp13, YRp7, YCp50 etc. can be illustrated.
In addition, the cells to be used as mammalian cells or insect cells include p91023, pCDM8, pcDL-SRα296, pBCMGSNeo, pSV2dhfr, pSVdhfr, pAc373, pACYM1, pRc / CMV, pREP4, pcDNA1, pVL1392 / 1393-AcHLT. / B / C and the like can be exemplified.
In addition to these existing gene expression vectors, it is of course possible to use gene expression vectors appropriately produced according to the purpose.
As a method for introducing a gene expression vector incorporating this chimeric gene into a host cell and transforming the host with this vector, for example, when the host cell is Escherichia coli or Bacillus subtilis, calcium chloride is used. When the host cell is a mammalian cell or an insect cell, a calcium phosphate method, an electroporation method, a liposome method, or the like can be selected.
The chimeric protein is accumulated in the culture system by culturing the transformant thus obtained according to a conventional method.
The medium used in such culture can be appropriately selected according to the type of the selected host. For example, when the host is Escherichia coli, an LB medium, TB medium or the like can be appropriately selected, and when the host is a mammalian cell, an RPMI 1640 medium or the like can be selected as appropriate.
Isolation and purification of the chimeric protein from the culture obtained by culturing the transformant as described above can be performed according to a conventional method. For example, the culture is treated with physical properties of the chimeric protein. And / or various processing operations utilizing chemical properties.
Specifically, for example, treatment with a protein precipitant, ultrafiltration, gel filtration, high performance liquid chromatography, centrifugation, electrophoresis, affinity chromatography using specific antibodies, dialysis, etc., alone or in combination, The chimeric protein can be isolated and purified.
As described above, the present chimeric gene and the present chimeric protein can be obtained. This chimera gene is the basis for producing this chimera protein. The present chimeric protein can also be used as an immunogen for antibodies used for carrying out this detection method. Further, the present chimeric gene and chimeric protein can be used in screening methods for developing therapeutic agents for ALL, which will be described later.
2. Antibodies against this chimeric protein
Depending on the mode of the present detection method, it is necessary to use an antibody against the present chimeric protein.
As described above, the chimeric protein to be detected is a fusion protein of a part of MEF2D protein and a part of DAZAP1 protein. As an antibody that can be used in the present detection method, 1) the present chimeric protein An antibody specific for a part of MEF2D protein and a part of DAZAP1 protein, or 2) an antibody specific for only a part of MEF2D protein and only a part of DAZAP1 A specific set of antibodies may be mentioned.
These antibodies can be obtained by selecting antibodies having the desired properties from antibodies produced using the present chimeric protein as an immunogen, but the antibodies are produced in advance by limiting the immunogen to a specific form. Is efficient.
That is, the immunogens of an antibody specific for only a part of MEF2D protein and an antibody specific for only a part of DAZAP1 protein are the chimeric protein (MEF2D-DAZAP1 protein and DAZAP1-MEF2D protein, respectively). ) Is preferred.
On the other hand, an antibody specific for a part of MEF2D protein and a part of DAZAP1 protein constituting the present chimeric protein is at least a part of MEF2D protein constituting the present chimeric protein and DAZAP1 as an immunogen. An oligopeptide containing a part of the protein binding part (for example, the glycine at position 222 for an antibody against the chimeric protein of SEQ ID NO: 5 and the 155th glycine for an antibody against the chimeric protein of SEQ ID NO: 6) Is preferred. The chain length of the oligopeptide is preferably about 20 to 50 amino acids.
When the antibody against the present chimeric protein is a polyclonal antibody, it can be produced from immune serum derived from an animal immunized with the above immunogen (including those conjugated with a hapten such as umbrella hemocyanin if necessary). it can.
In addition, a cell line derived from an animal of the same type and strain as the immunized animal is transformed by introducing an expression vector incorporating the chimeric gene or a gene encoding a part thereof, and the transformed cell is transformed into the cell line. The desired polyclonal antibody can be prepared by transplanting to an immunized animal. That is, the chimeric protein is continuously produced from the transformed cell in the body of the animal transplanted with the transformed cell, and an antibody against it is produced, which can be used as a target polyclonal antibody (Nemoto). , T., et al., Eur. J. Immunol., 25 3001 (1995)).
Furthermore, by administering the expression vector expressing the chimeric gene directly to the animal by means such as intramuscular injection or subcutaneous injection, the chimeric protein is continuously produced in the animal and the transformed cells are transplanted. In the same manner as described above, the desired polyclonal antibody can be produced (Raz, E., el al., Proc. Natl. Acad. Sci. USA, 91 9519 (1994)).
On the other hand, a monoclonal antibody against the chimeric protein is an antibody that recognizes the chimeric protein by producing a hybridoma between the immune cells of the immunized animal and the myeloma cells of the animal in the same manner as the above polyclonal antibody. Can be produced by selecting a clone that produces and culturing this clone.
Animals to be immunized to produce antibodies against this chimeric protein are not particularly limited, and mice, rats, etc. can be widely used. When producing monoclonal antibodies, bone marrow used for cell fusion is used. It is desirable to select in consideration of compatibility with tumor cells.
Immunization can be performed by a general method, for example, by administering the immunizing antigen to an animal to be immunized by intravenous, intradermal, subcutaneous, intraperitoneal injection or the like.
More specifically, the immunizing serum or monoclonal for the production of polyclonal antibodies is administered to the animal to be immunized several times by the above means every 2 to 14 days in combination with a normal adjuvant if desired. Immune cells for antibody production such as spleen cells after immunization can be obtained.
In the case of producing a monoclonal antibody, myeloma cells as the other parent cells to be fused with the immune cells are already known, for example, SP2 / 0-Ag14, P3-NS1-1-Ag4-1, MPC11- 45,6. TG 1.7 (from the mouse); 210. RCY. Ag1.2.3 (rat origin); SKO-007, GM 15006TG-A12 (above, human origin), etc. can be used.
Cell fusion between the above immune cells and the myeloma cells can be performed by a generally known method such as the method of Kohler and Milstein (Kohler, G. and Milstein, C., Nature, 256 , 495 (1975)).
More specifically, this cell fusion is carried out by adding an auxiliary agent such as dimethyl sulfoxide in order to improve the fusion efficiency in the presence of a commonly known fusion promoter such as polyethylene glycol (PEG), Sendai virus (HVJ), etc. Hybridomas are prepared by carrying out in a normal culture medium added as necessary.
Separation of a desired hybridoma can be performed by culturing in a normal selection medium such as HAT (hypoxanthine, aminopterin and thymidine) medium. That is, the hybridoma can be separated by culturing in this selection medium for a time sufficient for cells other than the target hybridoma to die. The hybridoma obtained in this way can be subjected to the search for a monoclonal antibody of interest and single cloning by the usual limiting dilution method.
The target monoclonal antibody-producing strain can be searched according to a general search method such as ELISA method, plaque method, spot method, pseudo-collection reaction method, octarony method, RIA method.
The hybridoma producing the desired monoclonal antibody that recognizes the present chimeric protein thus obtained can be subcultured in a normal medium, and can also be stored for a long time in liquid nitrogen.
Collection of the desired monoclonal antibody from this hybridoma can be achieved by culturing the hybridoma according to a conventional method and obtaining it as a culture supernatant, or by administering the hybridoma to an animal that is compatible with the hybridoma and proliferating it. A method for obtaining the ascites can be used.
It should be noted that immune cells are cultured in vitro in the presence of the present chimeric protein or a part thereof, and after a certain period of time, a hybridoma of the immune cells and myeloma cells is prepared using the cell fusion means, and an antibody-producing hybridoma is obtained. A desired monoclonal antibody can also be obtained by screening (Reading, CL, J. Immunol. Meth., 53 , 261 (1982); Pardue, R .; L. , Et al. , J .; Cell Biol. 96 1149 (1983)).
The polyclonal antibody and monoclonal antibody obtained above can be further purified by ordinary means such as salting out, gel filtration, affinity chromatography and the like.
The thus obtained polyclonal antibody and monoclonal antibody are antibodies having specific reactivity with the present chimeric protein.
The antibody thus obtained can be used after being labeled with a labeling substance as required in the detection means specifically used as described above.
Such a labeling substance is a labeling substance that provides a detectable signal by reacting the labeling substance alone or with another substance. Specifically, for example, horseradish peroxidase, alkaline phosphatase, β -Enzymes such as D-galactosidase, glucose oxidase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, malate dehydrogenase, penicillinase, catalase, apoglucose oxidase, urease, luciferase or acetylcholinesterase, fluorescein isothiocyanate, Fluorescent substances such as phycobiliprotein, rare earth metal chelate, dansyl chloride or tetramethylrhodamine isothiocyanate, ¹²⁵ 1, ¹⁴ C, ³ Examples thereof include radioisotopes such as H, chemical substances such as biotin, avidin and digokesigenin, and chemiluminescent substances.
As an antibody labeling method using these labeling substances, a known labeling method can be appropriately used according to the type of labeling substance to be selected.
Further, the present detection method can be used as an immobilized monoclonal antibody in which a monoclonal antibody (including a labeled one) against the present chimeric protein is immobilized on an insoluble carrier.
As such an insoluble carrier, various insoluble carriers already used as an insoluble carrier for antibodies can be used. Specifically, for example, a plate represented by a microplate, a test tube, a tube, a bead, a ball, a filter, a membrane, or a cellulose carrier, an agarose carrier, a polyacrylamide carrier, a dextran carrier, a polystyrene carrier, Examples include insoluble carriers used in affinity chromatography such as polyvinyl alcohol carriers, polyamino acid carriers, and porous silica carriers.
As the method for immobilizing monoclonal antibodies on these insoluble carriers, the antibody immobilization methods already established on various insoluble carriers can be appropriately selected according to the type of insoluble carrier to be selected.
Thus, a monoclonal antibody against the present chimeric protein that can be used in the present detection method can be prepared.
3. This detection method
As described above, the present detection method is a method for detecting a chimeric protein, wherein the precursor B cell ALL of the sample provider is discriminated by detecting the present chimeric gene or the present chimeric protein in the sample. In any of the detection methods, the sample provider in which the present chimeric protein is detected in the sample may have different symptoms, treatment course, and prognosis as compared to the precursor B cell ALL with other types of chromosome translocation. High nature.
1) Sample
The sample to be detected by this detection method is a sample derived from a leukemia patient whose ALL should be determined. Specifically, a blood sample, a buffy coat, or the like can be used. As for the blood sample, a known blood cell fraction sample can be prepared by subjecting a blood cell fraction prepared by performing a centrifugal separation process or the like to a known treatment. In this detection method, it is preferable to use a leukocyte fraction specimen.
The blood for obtaining this blood cell fraction sample is not particularly limited, and may be arterial blood or venous blood. Generally, peripheral blood is used because of its ease of collection.
2) A mode for detecting this chimeric protein
This embodiment is an embodiment in which the present chimera protein is detected in a specimen mainly using an antibody against the chimera protein described above. Specific examples of detection means include enzyme immunoassay, radioimmunoassay, analysis by flow cytometry, Western blot, immunoprecipitation / immunoblot, immunocytochemical staining, and the like.
First, the enzyme immunoassay method is also referred to as an enzyme immunoassay method, and is a detection method using an enzyme as a labeling substance in a labeled immunoassay method (a detection method using a radioisotope is a radioimmunoassay method). Enzyme immunoassay methods are roughly classified into “heterogeneous enzyme immunoassay” requiring B / F separation and “homogeneous enzyme immunoassay” not requiring this B / F separation.
In the present detection method, it is more preferable to select the former “heterogeneous enzyme immunoassay”, which is generally said to have high measurement sensitivity, and to select “enzyme-linked immunosorbent assay (ELISA)” using an immunosolvent. More preferably.
As a more specific detection mode, an enzyme immunoassay method (hereinafter also referred to as a sandwich method) by a so-called sandwich method can be exemplified.
Such a sandwich method is one of particularly preferable detection modes particularly considering the convenience of operation and the convenience of economy, especially the versatility as a clinical test.
In carrying out this sandwich method, an antibody against the present chimeric protein is immobilized on a microplate having a large number of wells such as a 96-well microplate, and the horseradish peroxidase described above. It is preferable to use an antibody against the present chimeric protein labeled with an enzyme such as the above or biotin.
The sandwich method is an enzyme immunoassay method including at least the following steps (a) and (b).
(A) A step of reacting an immobilized antibody obtained by immobilizing an antibody against this chimeric protein on an insoluble carrier with granulocyte lysate prepared from a blood sample.
In this step (a), the microplate used is usually washed after the reaction, and the unreacted specimen is removed from the immobilized monoclonal antibody.
(B) The present chimeric protein labeled with horseradish peroxidase or biotin on an antigen-antibody complex formed by binding of the immobilized monoclonal antibody to the MEF2D-DAZAP1 chimeric protein and DAZAP1-MEF2D fusion protein in the sample Reacting an antibody against.
In this step (b), usually, after the reaction, the used microplate is washed, and the unreacted labeled antibody is removed from the immobilized antibody.
Further, in this step (b), it is necessary to reveal the label signal by using a means for expressing the label signal according to the type of the labeling substance in the reacted second antibody. For example, when biotin is used as the labeling substance, the labeling signal can be revealed using avidin or the like. For example, when horseradish peroxidase is selected as a labeling substance, the substrate can be added together with a coloring substance as necessary to reveal the labeling signal.
Thus, the present chimeric protein in a desired sample can be detected by detecting the developed color signal using a signal specifying means corresponding to the type of the color signal.
The present chimeric protein can be detected using immunocytochemical staining. A smear is prepared using a cell smear centrifuge Cytofuse 2 (Nippon Luft Co., Ltd.). The smear sample can be stained with the antibody against the chimeric protein by a conventional method to stain the chimera protein in the smear sample.
3) Aspects of methods for detecting the presence of mRNA expressed in a specimen
This embodiment is an embodiment of the present detection method for discriminating the precursor B cell ALL of the sample provider by detecting a gene encoding the present chimeric protein in the sample. Specific detection means include DNA chip method, Southern blot method, Northern blot method, Real-time RT-PCR method, Nested PCR method, Inverse PCR method, Nested Inverse PCR method, Invader method, FISH method, Comparative Genomic Hybridization (CGH ) The law etc. can be illustrated.
A representative embodiment is that the gene of the present chimeric protein is expressed by detecting mRNA in the blood cell fraction indirectly by detecting cDNA in the sample using the mRNA of the present chimeric protein as a template. (Hereinafter, the detection means of this embodiment is also referred to as a chimeric mRNA detection method. The detection method of this embodiment includes the above-mentioned real-time RT-PCR method).
In the chimeric mRNA detection method, typically, mRNA is selected from the total RNA of the specimen according to a known method (for example, a method using oligo dT). Then, from the obtained mRNA, a gene amplification capable of amplifying a gene using mRNA as a template, using a heat-resistant DNA polymerase using a nucleotide chain corresponding to a known nucleotide sequence of the present chimeric gene as an amplification primer The presence of mRNA of the chimeric protein in a sample is preferably detected by amplifying cDNA encoding the chimeric protein by a method such as RT-PCR and detecting the presence or absence of the amplification product by the gene amplification procedure. Can be detected in real time.
Using the cDNA encoding the chimeric protein amplified as described above as a template, a gene amplification operation using a heat-resistant DNA polymerase such as PCR is further performed to detect the presence or absence of the gene amplification product. Thus, the presence of mRNA of the present chimeric protein in the sample can be detected more accurately (the gene amplification primer used in the second gene amplification operation is the gene amplification used in the first gene amplification operation). It is necessary to use a nucleotide chain corresponding to the inside of this chimeric gene as a primer for gene amplification rather than a primer for gene).
The invader assay method (Third Wave Technologies (USA)) hybridizes a template nucleotide chain (cDNA obtained from a specimen) with a first nucleotide chain (1: Invader Probe) having the following characteristics 1 and 2. After that, the second nucleotide strand (2: Signal Probe) is hybridized, and then the partial triplex structure of the nucleotide strand is allowed to act on its 3 ′ side with a nuclease that specifically cleaves (Cleavease). By detecting the detection portion of the second nucleotide chain cleaved by this nuclease, a translocation comprising a part of MEF2D mRNA and a part of DAZAP1 mRNA constituting the present chimeric protein in the template nucleotide chain Nucleotide encoding site A reaction based on a method for detecting a chain can be mentioned.
1. First nucleotide chain: a complementary nucleotide chain to a nucleotide chain encoding a translocation site composed of a part of MEF2D protein and a part of DAZAP1 protein constituting the chimeric mRNA.
2. Second nucleotide chain: a template nucleotide having a “complementary portion” complementary to the nucleotide chain encoding the translocation site on the 3 ′ side and provided with a detection element continuously therewith There is a “detection portion” that is non-complementary to the 5 ′ side, and the 5′-most base of the “complementary portion” is a base that is complementary to the base encoding the above-mentioned binding site. A complex nucleotide chain. A triple structure is formed by the template DNA, the first nucleotide chain and the second nucleotide chain, and this structure is recognized by Cleavease and cleaved.
The DNA chip method is a method for quantifying mRNA expressed in cancer (leukemia) cells. For example, a synthetic oligonucleotide having the above-mentioned binding site of the present chimeric gene is immobilized on a substrate (cDNA can also be immobilized), and labeling is performed when RNA prepared from a specimen is synthesized by reverse transcriptase. The expression level of mRNA can be measured by hybridizing the labeled cDNA and the synthetic oligonucleotide on the substrate and scanning the amount of bound label.
The Southern blotting method isolates and immobilizes the genomic DNA obtained from the specimen, and the Northern blotting method isolates and immobilizes the mRNA, and then detects hybridization with the binding site of the chimeric gene, thereby detecting the chimera in the specimen. This is a method for detecting the presence of a gene.
4) A mode for detecting genomic DNA present in a specimen
As a representative method of this embodiment, CGH (Comparative Genomic Hybridization) method and FISH method [Fluorescence in situ hybridization (FISH): Yasui, K. et al. Imoto, I .; , Fukuda, Y .; , Pimkahaokham, A .; Yang, Z .; Q. Naruto, T .; , Shimada, Y .; Nakamura, Y .; , And Inazawa: Identification of targets genes with an amplicon at 14q12-q13 in esophageal squamous cell carcinoma. Genes Chromosomes Cancer, 32 112-118, 2001]. In this detection method of this embodiment, RP11-98G7, which is a BAC clone having a translocation site of this chimeric gene, is labeled and, when FISH is performed, the translocation portions of chromosomes 1 and 19 that are reciprocal translocation sites are detected. Can do.
In this detection method, the pathological condition of ALL can be determined by detecting the present chimeric gene and protein in the specimen in this manner. It is to be noted that, by combining this detection method with an existing ALL pathological condition determination method, it is possible to more accurately determine the ALL pathological condition.
4). Screening method for anti-leukemia drugs
As described above, the present chimeric protein strongly interacts with MEF2 family proteins, which is likely to be associated with the development of leukemia. Such specific inhibitors of interaction can be expected as therapeutic agents for leukemia.
Therefore, the present invention is an invention that provides a method for screening an active ingredient of a therapeutic agent for leukemia (hereinafter also referred to as the present screening method) using the specific inhibitory effect of the above interaction as an index.
This screening method is specifically defined as the method shown below.
a) Quantifying the binding amount of both proteins in the presence of the MEF2 family protein and the present chimeric protein (MEF2D-DAZAP1 protein or DAZAP1-MEF2D protein)
b) In the quantification system of a), when the MEF2 family protein and the present chimeric protein are in contact with each other or when the test substance is allowed to coexist in advance, the binding amount of the two proteins is quantified,
c) A screening method for a test substance, wherein the test substance is screened as an anti-leukemia substance when the binding quantity in b) is smaller than the binding quantity between the MEF2 family protein and the chimeric protein in a) .
In the above screening method, examples of MEF2 family proteins include MEF2D protein, HDAC4 protein, and p300 protein. Among these, it is preferable to use MEF2D protein or HDAC4 protein. These MEF2 family proteins can be produced using a conventional method according to the production method of the fusion protein described above.
In the present screening method, the MEF2 family protein and the present chimeric protein coexist in the cell and the test substance is added to the outside of the cell, the binding between the actual MEF2 family protein and the present chimeric protein in vivo, It is possible and preferred to detect inhibition of such binding by medication. When this embodiment is used, it is preferable to transform a test cell with a vector incorporating a gene encoding a MEF2 family protein and a vector incorporating the present chimeric gene. The integration cells are not particularly limited, but preferably human cells, particularly cervical cancer (Hela) cells, leukemia (K562) cells, colon cancer (HT-29) cells, kidney cancer (HEK293) cells are used. Is preferred.
Quantification of the amount of bound protein can be performed by a conventional method that can be employed for the quantification of trace amounts of protein, and specific examples will be described later.
Further, as a premise for performing this screening method, it is preferable to perform primary screening. Specifically, for example, leukemia cells (for example, THP4 cells) that inhibit proliferation of leukemia cells (for example, TS-2 cells) in which MEF2D-DAZAP1 translocation has occurred but have E2A-PBX1 translocation have occurred. It is preferred to screen for substances that do not inhibit or only weakly inhibit the growth of the.
Further, the substance screened as an active ingredient of a desired therapeutic agent for leukemia by carrying out this screening method is further subjected to in vivo screening, for example, TS-2 cells in nude mice in which the above TS-2 cells are implanted. It is preferable that the final screening is performed by a screening method using as an index the growth-inhibiting effect and the survival rate of the nude mouse.
The present invention provides a screening kit for performing the screening method described above. The kit includes, as a minimum component, a vector incorporating a gene encoding a MEF2 family protein and a vector incorporating this chimeric gene. Furthermore, other elements necessary for carrying out the present screening method, for example, cells to be transformed with these vectors, a culture solution of the cells, a dilution buffer, a cell lysis buffer, a fusion protein, and It is also possible to add an element (for example, a specific antibody, an affinity chromatography column, etc.) for detecting free protein.

FIG. 1 is a drawing showing mapping of translocation points in the region of chromosome 1q21-q23 by FISH method and Southern blotting.
(A) Analysis by FISH method was performed using 10 types of BAC clones covering the region of chromosome 1q21-q23. Among them, BAC clone RP11-98G7 (indicated by open bars) is the chromosome of der (1) and der (19) of TS-2 cells (indicated by arrows in the right figure) and chromosome 1 (indicated by arrowheads in the right figure). ). RP11-98G7 is a clone having only the MEF2D sequence.
(B) In order to narrow the translocation point, a design was made based on the RP11-98G7 sequence, a probe was prepared by PCR, and a Southern blot analysis was performed. After digestion with EcoRI / HindIII or BglII / HindIII, DNA bands (arrow b and arrow d on the right panel) that were not detected in the genomic DNA of healthy subjects were found in genomic DNA derived from TS-2 cells. This DNA band was not found in THP-4 cells or normal peripheral blood leukocytes. Comprehensively judging these results and the results of Southern blot analysis after digestion of each enzyme alone, the translocation point is within a range of about 1.8 Kb from the EcoRI site of chromosome 1q22 based on the restriction enzyme map of chromosome 1q22. (Left panel). The black double arrows (a and c) and the gray double arrows (b and d) in the right panel respectively match the bands a to d detected by the Southern blot.
FIG. 2 is a photograph showing cloning of the gene sequence at the translocation point.
(A) Inverse-PCR method schema used to identify translocation points. Based on the predicted restriction enzyme map of the translocation region, genomic DNA was first digested with the restriction enzyme HindIII. Circular DNA produced by self-binding was purified, and PCR was performed using primers designed from BAC98G7 with a sequence close to the expected translocation point. The PCR product was subcloned and the nucleotide sequence was determined. A1 / A2 primers were used for the first PCR, B1 / B2 primers were used for the nested PCR, and C1 / C2 primers were used for the control PCR.
(B) A band of 1.6 Kb was detected when B1 / B2 was used as a primer by agarose gel electrophoresis after nested inverse PCR using the genome of TS2 cells as a template. On the other hand, this band was not detected when the genome of normal human peripheral blood leukocytes (indicated as N) was used. When primer C1 / C2 was used, a 0.9 Kb band was detected using both genomes as templates (control).
(C) When BLAST search was performed on the website “GenBank”, the sequence of the Inverse-PCR product was the chromosome 1q22 having the intron 6 of MEF2D, the chromosome 19p13.3 having the intron 6 of DAZAP1, and the sequence of the translocation point. there were. The partial sequence around the translocation point is described. The origin of the extra 5 bases shown in bold is unknown.
FIG. 3 is a photograph showing identification of MEF2D-DAZAP1 chimeric transcript by RT-PCR.
(A) RT-PCR to identify MEF2D-DAZAP1 and DAZAP1-MEF2D chimeric transcripts. The templates used were TS-2 cell genome (TS-2), healthy subject peripheral blood leukocyte genome (N) and THP-4 cell (THP-4) genome. MEF2D-DAZAP1 and DAZAP1-MEF2D chimeric transcripts were detected only in TS-2 cells. E2A-PBX1 chimeric transcripts, wild type MEF2D and wild type DAZAP1 transcripts were detected in THP-4 cells.
(B) Detection of chimeric transcripts in 2 leukemia clinical cases. MEF2D-DAZAP1 and DAZAP-MEF2D chimeric transcripts of the size found in the TS-2 cell genome were detected by RT-PCR using the patient 1-derived genome as a template. RT-PCR product of the same size as the E2A-PBX fusion transcript was detected in the precursor B cell ALL Patient 2 having the same chromosomal translocation t (1; 19) as that of Patient 1.
(C) Expected view of wild-type MEF2D protein, wild-type DAZAP1 protein, and predicted MEF2D-DAZAP1 and DAZAP1-MEF2D fusion protein production. The MEF2D protein has a MADS domain and a MEF2 domain at the N-terminus, and a transcription activation domain at the C-terminus. The DAZAP1 protein has two RNA recognition motifs (RRM) at the N-terminus. The chimeric MEF2D-DAZAP1 protein is composed of a C-terminal region partially including the MADS domain / MEF2 domain of the MEF2D protein and the second RRM domain of the DAZAP1 protein. The other DAZAP1-MEF2D fusion protein consists of an N-terminal region having one RRM domain of DAZAP1 and another incomplete RRM domain, and a C-terminal region of MEF2D having a transcriptional activation site.
FIG. 4 is a photograph showing the subcellular localization of a chimeric protein by fluorescence immunochemistry using a transiently expressed epitope tag protein.
Expression plasmids constructed by fusing cDNAs having the full length of MEF2D, DAZAP1, MEF2D-DAZAP1 and DAZAP1-MEF2D with FLAG tags were transiently transfected into cells. Each protein was detected using an anti-FLAG (M2) monoclonal antibody.
FLAG tag MEF2D and FLAG tag MEF2D-DAZAP1 showed a staining pattern scattered in the nucleus, and FLAG tag DAZAP1 and FLAG tag DAZAP1-MEF2D showed a more diffuse staining pattern in the nucleus.
FIG. 5 is a photograph showing the interaction between MEF2D-DAZAP1 and MEF2D or HDAC4.
(A) FLAG tag MEF2D and Myc tag MEF2D-DAZAP1 were expressed in HEK293 cells by transient cotransfection, and MEF2D protein was immunoprecipitated from the cell lysate using anti-FLAG antibody. It was shown that MEF2D protein and MEF2D-DAZAP1 protein co-precipitated.
(B) FLAG tag HDAC4 and Myc tag MEF2D-DAZAP or Myc tag MEF2D were transiently expressed in HEK293 cells. HDAC4 protein, MEF2D protein and MEF2D-DAZAP chimeric protein were co-precipitated using antibodies. It was shown that the interaction between MEF2D-DAZAP1 and HDAC4 is stronger than the interaction between MEF2D and HDAC4.

Identification of translocation site of chromosome 1q22 by fluorescence in situ hybridization method (FISH method) TS-2 cell, a pre-B cell ALL cell line, has chromosomal translocation t (1; 19) (q23; 13) Responsible. However, the E1A-PBX1 fusion gene resulting from the chromosomal translocation reported so far is not detected (Yoshinari, M., Imaizumi, M., Eguchi, M., Ogasawara, M., Saito, T., Suzuki, H., Koizumi, Y., Cui, Y., Sato, A., Saisho, T., Ichinohasayama, R., Matsubara, Y., Kamada, N. & Iinuma, K .: Establishment of a novel ( TS-2) of pre-B exact lymphophobic leukemia with at (1; l9) not involving the E2A gene, Cancer Genet. Cytogene., 01,95-102,1998). In order to solve this problem, it is necessary to determine an accurate translocation site and identify a fusion gene associated with the translocation.
First, in order to determine the translocation site, analysis was performed by the FISH method using 10 BAC clones having the chromosome 1q22-q23 region as probes. The 10 BAC clones were selected with reference to the National Center for Biotechnology Information and the University of California Santa Cruz Biotechnology database. Among the BAC clones used here, FSH of TS-2 cells was performed using RP11-98G7 (GenBank accession number AL139412) as a probe. As a result, der (1) and der (19) chromosomes that are reciprocal translocation sites. (Right diagram in FIG. 1A). Detailed analysis revealed that RP11-98G7 is composed of a part of the MEF2D genomic gene region present in the chromosome (1q22) and contains a translocation point of the MEF2D gene (FIG. 1A, left explanatory diagram). ). Furthermore, the region of the translocation site was clarified using Southern blot analysis. One type of probe in the RP-98G7 sequence detected a translocation site of TS-2 cells, but could not be detected in THP-4 cells isolated from normal ALL or normal peripheral blood leukocytes. Furthermore, the translocation region can be mapped by Southern analysis after restriction enzyme digestion and restriction enzyme site analysis using a computer of chromosome 1q22, and the translocation site is about 1.8 Kb from the EcoRI recognition site of chromosome 1q22. (FIG. 1B).

Identification of a gene localized at the translocation site of chromosome 19p13 by Inverse PCR method In order to clarify the translocation site, a primer designed in the chromosome 1q22 region was used to self-treat the HindIII-digested TS-2 cell genomic DNA. Inverse PCR was performed using the ligated circular DNA as a template (FIG. 2A). The product subjected to Nested-PCR was 1.6 Kb of the expected size and was specifically detected in TS-2 cells (FIG. 2B). When the nucleotide sequence was determined, this PCR product was found to have both the chromosome 1q22 and 19p13.3 sequences simultaneously with the translocation site sequence (FIG. 2C). This base sequence contained DAZAP1 (19p13.3) of the chromosome 19p translocation site. The translocation site spanned intron 6 of the MEF2D gene and intron 6 of the DAZAP1 gene. Furthermore, the rearranged genomic base sequence was determined by PCR using the genomic DNA of TS-2 cells as a template using primers specific to the MEF2D gene and the DAZAP1 site.

Identification of fusion transcript resulting from translocation using RT-PCR method After synthesizing cDNA from the fusion transcript resulting from translocation using reverse transcriptase and determining its nucleotide sequence, MEF2D gene It was found that exon 6 and exon 7 of the DAZAP1 gene were fused to generate MEF2D-DAZAP1 fusion mRNA. Since two pairs of chromosome translocations occurred, MEF2D-DAZAP1 and DAZAP1-MEF2D fusion products were predicted to be generated and detected. When RT-PCR was performed using mRNA prepared from TS-2 cells, two types of fusion mRNA could be detected in addition to wild-type MEF2D mRNA and DAZAP1 mRNA (FIG. 3A, column indicated by TS-2). Two types of cDNA detected here were cloned into a pCRII vector (Invitrogen, CA, USA), and their base sequences were determined. The amino acid sequences of both chimeric proteins were determined from the nucleotide sequence data. As a result, the MEF2D-DAZAP1 cDNA sequence (SEQ ID NO: 3), the amino acid sequence of the same protein (SEQ ID NO: 5), the DAZAP1-MEF2D cDNA sequence (SEQ ID NO: 4), and the amino acid sequence of the same protein (SEQ ID NO: 6) are determined. did. On the other hand, these fusion mRNAs were not detected from THP-4 cells (FIG. 3A, column labeled THP-4). Furthermore, E2A-PBX1 fusion mRNA detected in THP-4 cells was not detected in TS-2 cells.
Next, as a result of preparing RNA from bone marrow cells of a patient (Patient 1) from which TS-2 cells were isolated and attempting to detect a fusion transcript derived from a translocation site by RT-PCR, two types were expected as expected. Of fusion mRNA could be detected (FIG. 3B). The two fusion transcripts were not detected when RNA isolated from bone marrow cells of 13 ALL patients with other t (1; 19) translocations was used. As a result of determining the nucleotide sequence of the fusion transcript, fusion occurred so that the protein sequence of the downstream gene was translated. That is, it was found that the 222nd codon of the MEF2D gene and the 155th codon of DAZAP1 both encode glycine, and are fused in a state where the amino acid translation frames are matched to produce a chimeric transcription product.
A number of hematological cancer cases have been reported in ALL patients with (1; 19) (q23; p13) translocation but no chimeric gene with E2A detected (Privitera, E., Kamps, MP, Hayashi, Y., Inaba, T., Shapiro, L.H., Raimondi, SC, Behm, F., Hendershot, L., Carroll, A.J., Baltimore, D., & Look, A.T. .: Differential molecular consequences of the 1; 19 chromosomal translocation in childhood acculative lyphophobic leukemia, 88, 1981, 1981. Have been, they are likely to MEF2D-DAZAP1 fusion product has become the cause.
The MEF2D gene has approximately 34 Kb and consists of 12 exons (Breitbart, RE, Liang, CS, Smoot, LB, Laheru, DA, Mahdavi, V. & Nadal-Ginard , B .: Afour human MEF2 transcription factor, hMEF2D, is an early marker of the myogenic line. Development, 118, 1095-1106, 1993). It encodes a MADS-box family protein of transcription factors (FIG. 3C). This protein forms a homodimer or heterodimer and binds to a consensus sequence of the expression control region of a number of muscle-specific and growth-induced genes, and is involved in skeletal muscle gene and myocardial differentiation (Hobson, G. et al. M., Krahe, R., Garcia, E., Siciliano, MJ, & Funanage, V.L.:Regional chromosomal assignments for two tens of mad mess. , 19p12, 5q14 and 1q12-23q.Genomics, 29, 704-711, 1995; nsey, TA, Zhang, CL & Olson, EN: MEF2: a calcium-dependent regulator of cell division, differential and death. Trends Biochem., 27, 40-47). .
The DAZAP1 gene is composed of about 29 Kb and consists of 11 exons (Tsui, S. Dai, T., Roettger, S., Schempp, W., Salido, EC & Yen, PH: Identification of two novel proteins that interact with germ-cell-specific RNA-binding proteins DAZ and DAZL1, Genomics, 65, 266-273, 2000). The DAZAP1 protein has two RNA recognition motifs (RRM) and a proline-rich C-terminal region (FIG. 3C), and binds to DAZ and DAZL1 proteins via a DAZ repeat sequence (Dai, T., Vera, Y. et al. , Salido, E. C. & Yen, P. H .: Characterization of the mouse Dazap 1 gene encoding an RNA-binding protein with Lact. Since DAZAP1 is highly expressed in the testis, it is expected to play an important role in spermatogenesis (Vera, T., Dai, T., Hikim, AP, Lue, Y., Salido, E , Swedloff, R.S. & Yen, P.H .: Deleted in azoospermia associated protein, 1 shuttles, between nucleus and cytoplasm dur. The MEF2D-DAZAP1 chimeric protein found in the present invention has an N-terminal region of MEF2D protein (having MADS domain and MEF2 domain necessary for dimer formation) and a C-terminal region partially including the second RRM of DAZAP1 protein. It is fused. On the other hand, in the DAZAP1-MEF2D chimeric protein, the N-terminal region partially including the first RRM and the second RRM of the DAZAP1 protein and the C-terminal region including the transcription activation region of the MEF2D protein are fused (FIG. 3C). .

Intracellular localization site of chimeric transcript MEF2D protein is a transcription factor having a DNA binding site and a transcription activation site (Breitbart, RE, Liang, CS, Smoot, LB, Laheru, D. et al.). . A., Mahdavi, V & Nadak -Ginard, B:. a fourth human MEF2 transcription factor, hMEF2D, is an early marker of the myogenic lineage, Development, 118, in 1095-1106,1993), DAZAP1 the RNA-binding protein (Tsui, S., Dai, T., Roetter, S., Schempp, W., Salido, E. C. & Yen, P. H .: Identification. f two novel proteins that interact with germ -cell-specific RNA-binding proteins DAZ and DAZ1, Genomics, 65, 266-273,2000). The intracellular localization site of the chimeric transcript was determined and compared with the localization of wild type MEF2D protein and wild type DAZAP1 protein. The amino acid sequence of the fusion protein product MEF2D-DAZAP1 is shown in Sequence 2, and the amino acid sequence of DAZAP1-MEF2D is shown in Sequence 2. In order to perform detection using a FLAG antibody, HeLa cells were transfected with an expression plasmid in which a FLAG tag was attached to the N-terminus of each protein and allowed to express transiently. As a result of fluorescent immunocell staining using an antibody against FLAG, FLAG tag MEF2D and FLAG tag MEF2D-DAZAP1 were localized in the nucleus and showed scattered staining. On the other hand, FLAG tag DAZAP1 and FLAG tag DAZAP1-MEF2D were also localized in the nucleus, but showed more diffuse stained images (FIG. 4).

Formation of heterodimer of MEF2D protein and MEF2D-DAZAP1 chimeric protein MEF2D is one of MEF2 family proteins such as HDAC4 and p300 protein, and MADS domain and MEF2 required for homodimer and heterodimer formation. Contains the domain (Figure 3C). Since the chimeric protein MEF2D-DAZAP1 contains MADS and MEF2 domains, it is thought to form a dimer with MEF2D itself or with other proteins that interact with MEF2D.
To prove this possibility, HEK293 cells were co-transfected with FLAG tag MEF2D and a plasmid expressing Myc tag MEF2D-DAZAP1, cell extracts were prepared and immunoprecipitated. As a result, since MEF2D and the chimeric protein co-precipitated (FIG. 5A), it is concluded that both of them form a complex in the cell. Similar results were obtained when the reverse combination, ie, Myc tag MEF2D-DAZAP1 and FLAG tag MEF2D expression plasmid were co-transfected. Furthermore, both proteins co-precipitated in the same manner when Myc tag MEF2D-DAZAP1 and FLAG tag HDAC4 expression plasmid were cotransfected.
Judging from the density of the band when coprecipitated, the MEF2D-DAZAP1 fusion protein binds to HDAC4 more strongly than MEF2D (FIG. 5B). When the Myc tag MEF2D-DAZAP1 and the FLAG tag HDAC4 expression plasmid were co-transfected into HeLa cells, it was revealed from the results of fluorescent immunocytochemistry that both proteins were present at the same site in a state of being scattered in the nucleus. This result is presumed that the MEF2D-DAZAP1 chimeric protein suppresses the function of MEF2D, possibly by forming a heterodimer with MEF2D.
So far, the property of transforming cells in the MEF2D-DAZAP1 and DAZAP1-MEF2D fusion proteins is not clear. However, the formation of a heterodimer with the intracellular localization site of both fusion proteins strongly suggests that the present chimeric protein may interact with MEF2 family proteins (such as MEF2D or HDAC4).
The role of MEF2 protein is usually limited to muscle tissue (Yu, YT, Breitbart, R., Smoot, LB, Lee, Y., Mahdavi, V. & Nadal-Ginard, B .: Human Myocyte-specific enhancer factor 2 complicates a group of tissue-related MADS box transcription factors., Genes Dev., 6 , et al., 1983D -Jun promoter is controlled (Han, TH & Prywes, R .: Regulatory role of MEF2D in serum induction of the -Jun promoter, Mol.Cell Biol., 15 , 2907-2915,1995), other tissue or cell differentiation, also likely to be involved in the proliferation.
The expression of MEF2A and MEF2D was remarkably increased in the process of differentiation of whole myeloid leukemia cells HL-60 into monosites (Shin, HH, Seoh, JY, Chung, HY, Choi). , SJ, Hahn, MJ, Kang, JS, Choi, MS & Han, TH: Requirements of MEF2D in the induced difference of HL60 promeloidol. , 36 , 1209-1214, 1999). Furthermore, when HL-60 cells expressed a dominant negative that does not contain the transcriptional active region of MEF2D, differentiation into monosites was suppressed. These suggest that the regulatory role of the MEF2 protein is widespread, and that MEF2D is required for the process of cell lineage differentiation rather than muscle tissue.
Therefore, it can be concluded that this chimeric protein is a fusion protein related to canceration.

Example of Screening System for MEF2D-DAZAP1 Chimeric Protein Inhibitor (a) Primary Screening :
As a primary screen, inhibits proliferation of leukemia (TS-2) cells in which the MEF2D-DAZAP1 translocation occurs, but does not inhibit or weaken the proliferation of leukemia (THP4) cells in which the E2A-PBX1 translocation occurs. Screen for substances that only inhibit. A 96-well microtiter plate was used for the method. TS-2 cells and THP4 cells were cultured at 37 ° C. in a CO ₂ incubator in the presence of RPMI-1640 medium containing 10% Fetal Bovine Serum, 100 units / ml penicillin and 100 μg / ml streptomycin. Incubate at At the time of culture, a compound having a concentration of 10 μM is added and cultured for 2 to 3 days. After culture, cell proliferation is measured by MTT assay. One example of an MTT assay reagent is commercially available as Cell Counting Kit-8 from Dojindo Laboratories, Inc. (Kumamoto). Compounds with an IC50 for TS-2 cell proliferation that is ¼ or less compared to the IC50 of THP4 cells are selected.
(B) Secondary screening (this screening method) :
Next, using HEK293 cells, a substance that inhibits the binding between this chimeric protein and the MEF2D protein is searched.
(1) The full-length Myc tag MEF2D-DAZAP1 gene and wild-type FLAG tag MEF2D gene are inserted into a pCMV-Tag2 or pCMV-Tag3 expression plasmid (Stratagene, CA, USA) so that the translation frame matches. After transformation of E. coli strain DH-5, a plasmid is prepared, and the target clone is selected by restriction enzyme mapping and nucleotide sequence determination.
(2) The FLAG tag MEF2D expression plasmid and the Myc tag MEF2D-DAZAP1 expression plasmid are co-transfected into HEK293 cells. Using FuGENE6 (Roche, Tokyo) as a transfection reagent, experiments can be performed according to the manual. After culturing in the above medium at 37 ° C. in a CO ₂ incubator for 1 day, the compound selected in the primary screening is added at a concentration of 10 μM and further cultured for 1 day.
(3) A HEK293 cell lysate is prepared by treatment with a TNE buffer (Kishida Chemical, Osaka) containing a protease inhibitor (Roche, Tokyo). Next, it is incubated at 4 ° C. with anti-Myc antibody (Cell Signaling Technology) and Protein G Sepharose beads for 2 hours. After separating the beads by centrifugation, the beads are washed twice with a TNE buffer containing a protease inhibitor and suspended in the loading buffer. The sample is heated at 100 ° C. for 5 minutes, and then subjected to SDS-PAGE electrophoresis. It was transferred to a nitrocellulose membrane, subjected to blocking treatment with Tris-buffered saline containing 0.05% Tween 20 and 5% nonfat dry milk, and incubated with anti-FLAG monoclonal antibody M2 (Sigma, MO, USA). The FLAG tag MEF2D protein is detected using SuperSignal West Dura Western blotting kit (Pierce Chemical CO., IL, USA).
As a result, a substance that inhibits the binding between the MEF2D-DAZAP1 chimeric protein and the MEF2D protein can be selected.
Next, a FLAG tag HDAC4 expression plasmid is prepared in the same manner. HEK293 cells are cotransfected with the FLAG tag HDAC4 expression plasmid and the Myc-tagged MEF2D-DAZAP1 expression plasmid in the same manner as described above. A substance that inhibits the binding between HDAC4 and MEF2D-DAZAP1 is identified using the same method as described above.
(C) Tertiary screening :
The compound thus obtained is an inhibitor of MEF2D-DAZAP1 chimeric protein, and in the next step, it can be tested whether the inhibitor is effective in a leukemia (TS-2) cell transplantation experiment using nude mice.

MEF2D-DAZAP1 Chimeric Protein Inhibitor Screening Method Using Homogeneous Time-Resolved Fluorescence Assay Method MEF2D-DAZAP1 chimeric protein, GST-MEF2D protein, and GST-HDAC protein are prepared as recombinant proteins using conventional methods. The MEF2D-DAZAP1 chimeric protein is labeled with biotin using a conventional method. Biotin-labeled (Bio-) MEF2D-DAZAP1 chimeric protein and GST-MEF2D protein are added in a buffer solution or appropriately diluted HEK293 extract and incubated at room temperature or 37 ° C. for 30 minutes. The binding of both proteins is detected by adding Europium Cryptate (EuK) labeled GST antibody (Packard, CT, USA) and Streptavidin (SA) labeled XL665 protein (Packard, CT, USA). Detection is performed using Discovery HTRF Microplate Analyzer (Packard, CT, USA). When irradiated with a laser beam of 337 nm, Europium Cryptate absorbs light of this wavelength and transfers the absorbed energy to the XL665 protein (acceptor). The XL665 protein emits 665 nm emission light, and this fluorescence intensity attenuates in a short time. In this reaction, Doner Fluorophore is EuK and the acceptor is an allophycocyanin protein XL665 which is stabilized. Fluorescence resonance energy transfer (FRET) catalyzed by both materials causes energy transfer with 50% efficiency when both are present at a distance of 9.5 nm.
A complex of EuK-GST antibody-GST-MEF2D-BioMEF2D-DAZAP1-SA-XL665 is formed, and the binding of GST-MEF2D and BioMEF2D-DAZAP1 chimeric protein is measured using FRET of EuK and XL665.
Compound that inhibits complex formation of both proteins in a high-throughput manner easily by incubating biotin-labeled MEF2D-DAZAP1 fusion protein and GST-MEF2D protein in the presence of 10 μM compound using a 96-well or 384-well microtiter plate Can be screened. This method is a homogeneous method and has a great feature in that it does not require B / F separation. The compound obtained here is further checked whether it inhibits the binding of biotin-labeled MEF2D-DAZAP1 fusion protein and GST-HDAC4 protein.
The compound selected here can be advanced to the secondary screening of (Example 6) and further to the tertiary screening to find the target drug.

  According to the present invention, a useful novel chimeric protein related to leukemia and a gene encoding the same are provided. According to the present invention, it has become possible to molecularly diagnose a translocation that has been unknown so far in the precursor B cell ALL. In other words, when the patient has a chromosomal translocation of t (1; 19) (q23; p13), only the generation of the E2A-PBX1 fusion gene could be detected so far. For the first time, gene generation can be monitored. This detection method can be performed by detecting a fusion gene, fusion mRNA, fusion protein, etc. on the genome using leukocytes, genomic DNA, cell lysate, RNA and the like prepared from the blood of the patient. Furthermore, a novel leukemia screening method was provided using this chimeric protein.

Claims (18)

A chimeric protein comprising a part of a MEF2D protein having the amino acid sequence shown in SEQ ID NO: 5 or 6 and a part of a DAZAP1 protein. A gene encoding the chimeric protein according to claim 1. The gene according to claim 2, wherein the gene encoding the chimeric protein having the amino acid sequence shown in SEQ ID NO: 5 is the gene having the base sequence shown in SEQ ID NO: 3. The gene according to claim 2, wherein the gene encoding the chimeric protein having the amino acid sequence shown in SEQ ID NO: 6 is the gene having the base sequence shown in SEQ ID NO: 4. An antibody against the chimeric protein according to claim 1 and specific for a part of MEF2D protein and a part of DAZAP1. 6. The antibody according to claim 5, wherein the antibody is a monoclonal antibody. The antibody according to claim 5, wherein the antibody is a polyclonal antibody. A set of antibodies specific for part of the MEF2D protein and antibodies specific for part of the DAZAP1 protein. A method for detecting a chimeric protein, wherein the precursor protein B acute lymphoblastic leukemia of the sample provider is discriminated by detecting the chimeric protein according to claim 1 in the sample. In the method for detecting a chimeric protein, the chimeric protein in a specimen is detected using an enzyme immunoassay method, a radioimmunoassay method, a Western blot method, an immunoprecipitation / immunoblot method, or an immunocytochemical staining method. 10. A method for detecting a chimeric protein according to item 9. A method for detecting a gene, wherein a precursor B cell acute lymphoblastic leukemia of the sample donor is discriminated by detecting a gene encoding the chimeric protein according to claim 2 in the sample. The method for detecting a gene according to claim 11, wherein the gene is genomic DNA, cDNA, or mRNA. In a method for detecting a gene encoding a chimeric protein, the gene in a sample is detected using a DNA chip method, a Southern blot method, a Northern blot method, a real-time RT-PCR method, an invader method, a FISH method, or a CGH method. The method for detecting a gene according to claim 11. The detection method according to any one of claims 9 to 13, wherein the sample is a blood sample. a) coexisting with the MEF2 family protein and the chimeric protein to quantify the binding amount of both proteins; b) in the quantitative system of a), when the MEF2 family protein and the chimeric protein contact or in advance Quantifying the binding amount of the two proteins when coexisting, and c) when the binding amount in b) is less than the binding amount of the MEF2 family protein in the a) and the chimeric protein, A screening method for a test substance, wherein the test substance is screened as an anti-leukemia substance. The screening method according to claim 15, wherein the MEF2 family protein is a MEF2D protein or an HDAC4 protein. The screening method according to claim 15 or 16, wherein the MEF2 family protein and the chimeric protein are allowed to coexist in the cell, and the test substance is added to the outside of the cell. A screening kit for use in the screening according to claim 17, comprising a vector incorporating a gene encoding a MEF2 family protein and a vector incorporating the chimeric gene.

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