JP5892666B2 - Cell death promoter - Google Patents

Description

  The present invention relates to a cell death promoting agent capable of promoting cell death by a synergistic effect with various drugs or various stimuli.

  Cancer is a disease that occupies most of the causes of death in humans, and conventionally, development of drugs used for cancer treatment has been vigorously advanced.

  There are various types of drugs used to treat cancer that kill cancer cells by various mechanisms.

  For example, drugs that kill cancer cells by inhibiting DNA synthesis, drugs that kill cancer cells by inhibiting cell division, drugs that kill cancer cells by damaging DNA (chromosomes), cancer cells There are drugs that kill cancer cells by antagonizing their metabolism, drugs that kill cancer cells by inhibiting nutrition, and the like (see Non-Patent Documents 1 and 2).

  However, the drugs used for conventional cancer treatment are often toxic themselves, and therefore often cause serious side effects in patients to which the drugs are administered.

  In order to suppress the side effects of conventional drugs, it is conceivable to reduce the amount of drugs administered to patients. However, in this case, side effects can be suppressed, but cancer cells cannot be effectively killed, resulting in a problem that the therapeutic effect is reduced.

  Therefore, at present, attention is focused on the development of drugs that have few side effects and can effectively kill cancer cells.

N J Curtin, “Inhibiting the DNA damage response as a therapeutic manoeuvre in cancer” British Journal of Pharmacology, (2013), 169, 1745-1765 Khurum H. Khan et al., “Cancer therapeutics: Targeting the apoptotic pathway”, Critical Reviews in Oncology / Hematology, (2014), http://dx.doi.org/10.1016/j.critrevonc.2013.12.012

  As described above, although attention has been focused on the development of drugs that have few side effects and can effectively kill cancer cells, such drugs have not yet been developed.

  If we can develop drugs that have few side effects and can effectively kill cancer cells, we will be able to save a huge number of patients suffering from cancer and contribute greatly to the medical field. Can do.

  Moreover, it is expected that the drug as described above functions as a drug capable of specifically killing not only cancer cells but also desired cells.

  For example, in fields such as molecular biology, the function of specific killed cells can be elucidated by killing only specific cells in a cell population and observing events that occur in subsequent cell populations There is. Therefore, if we can develop drugs that have few side effects and can effectively kill cancer cells, we can make a significant contribution not only to the medical field but also to academic fields such as molecular biology. it can.

  The present invention has been made in view of the above-mentioned conventional problems, and an object of the present invention is to provide a cell death promoter that has few side effects and can effectively kill desired cells. .

  In the process of studying the cell cycle, the present inventors pay attention to the binding of cyclin and the B′γ subunit of PP2A (Protein Phosphatase 2A), and kill the cell if the binding can be inhibited. We proceeded with further research based on our own hypothesis that it might be possible.

  Then, it was found that if a substance that inhibits the binding between cyclin and the B′γ subunit of PP2A (Protein Phosphatase 2A) is used, cells damaged by DNA can be effectively killed, and the present invention has been completed. I came to let you.

  In order to solve the above problems, the cell death promoting agent of the present invention comprises a protein containing at least a part of an ELA motif or a nucleic acid containing at least a part of a polynucleotide encoding the protein. It is characterized by being.

The cell death promoting agent of the present invention contains, as at least a part, a protein containing at least a part of the polypeptide described in (a) or (b) below, or a polynucleotide encoding the above protein. It preferably contains a nucleic acid. That means
(A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2;
(B) It consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 or 2, and inhibits the binding between cyclin and PP2A B′γ subunit. Polypeptide.

  The cell death promoting agent of the present invention is preferably administered to a living body that has developed DNA double-strand breaks or a living body that is subjected to treatment for causing DNA double-strand breaks.

  In the cell death promoting agent of the present invention, the living body is γ-irradiated, X-ray irradiated, administration of a topoisomerase I inhibitor (eg, camptothecin administration, irinotecan administration), or administration of a topoisomerase II inhibitor (eg, etoposide administration). ) Is preferably a living body that develops DNA double-strand breaks or a living body that is subjected to treatment for causing DNA double-strand breaks.

  The cell death promoting agent of the present invention preferably contains an agent that causes DNA double strand breakage.

  In the cell death promoting agent of the present invention, the drug is preferably at least one selected from the group consisting of a topoisomerase I inhibitor (for example, camptothecin, irinotecan) and a topoisomerase II inhibitor (for example, etoposide).

In the cell death promoting agent of the present invention, the polynucleotide encoding the protein is preferably a polynucleotide containing at least a part of the following DNA (c) or (d). That means
(C) DNA consisting of the base sequence shown in SEQ ID NO: 3 or 4;
(D) hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 3 or 4 and binding between cyclin and PP2A B′γ subunit DNA encoding a polypeptide that inhibits.

  In the cell death promoting agent of the present invention, the nucleic acid containing at least a part of the polynucleotide encoding the protein is one in which the polynucleotide encoding the protein is inserted into an expression vector. Is preferred.

  In the cell death promoter of the present invention, the expression vector is a plasmid, a phage vector, a viral vector (for example, a herpes virus vector, an adenovirus vector, an adeno-associated virus vector, and a retrovirus vector), or a virus particle. It is preferable.

  The cell death promoter of the present invention is preferably one that induces apoptosis.

  The present invention has an effect of allowing specific and efficient killing of cells that are damaged by DNA (for example, DNA double-strand breaks).

  Anticancer drugs are diverse and kill cancer cells by various mechanisms. For example, some anticancer agents kill cancer cells by inflicting damage (eg, DNA double strand breaks) on the DNA of the cancer cells. When such an anticancer agent and the cell death promoting agent of the present invention are used in combination, cancer cells damaged by DNA can be specifically and efficiently killed.

  The present invention has an effect that a cell death promoting agent that has very few side effects and is safe for a living body can be realized.

  Specifically, in the present invention, a polypeptide that has extremely few side effects and is safe for the living body (specifically, ELAS1 and ELAS2 described in the Examples) is used. The effect that an objective composition can be implement | achieved is produced.

  The cell death promoting agent of the present invention may inhibit the function of cyclin G1 or cyclin G2. However, cyclin G1 knockout mice, cyclin G2 knockout mice, and cyclin G1 and cyclin G2 double knockout mice are all born normally and grow healthy. In addition, these knockout mice are less likely to cause liver cancer even when administered with a chemical carcinogen compared to wild-type mice. This indicates that the cell death promoting agent of the present invention is safe for the living body even if the cell death promoting agent of the present invention inhibits the function of cyclin G1 or cyclin G2.

  Furthermore, since the present invention can increase the sensitivity of a living body to a drug (for example, a drug that generates DNA double-strand breaks), the effect of the drug can be sufficiently exerted with a small amount of the drug. In other words, the present invention can reduce the amount of drug to be used, and thus can prevent side effects due to the drug from occurring in the living body.

  For example, many anticancer agents currently used have serious side effects. In order to reduce the side effects, it is necessary to reduce the amount of the drug administered to the living body. However, if the amount of the drug administered to the living body is decreased, the anticancer effect is lowered. In this case, if the cell death promoter of the present invention is used, both reduction of side effects and sufficient anticancer effect can be realized simultaneously.

It is a figure which shows the structure of various polypeptide used for the Example of this invention. It is a figure which shows the result of the test for determining a binding domain in the Example of this invention. It is a figure which shows the result of the test for determining a binding domain in the Example of this invention. It is a figure which shows the result of the binding inhibition test in the Example of this invention. It is a figure which shows the preparation strategy of a knockout mouse in the Example of this invention. In the Example of this invention, it is a figure which shows the tumor produced in the liver of a knockout mouse. In the Example of this invention, it is a figure which shows the characteristic of the tumor which arose in the liver of a knockout mouse. It is a figure which shows the result of the test regarding the side effect of ELAS1 and ELAS2 in the Example of this invention. It is a figure which shows the result of the induction | guidance | derivation test of the apoptosis by a gamma ray in the Example of this invention. It is a figure which shows the result of the induction | guidance | derivation test of the apoptosis by the chemical | medical agent in the Example of this invention. It is a figure which shows the result of the induction | guidance | derivation test of the apoptosis by the chemical | medical agent in the Example of this invention. It is a figure which shows the result of the induction | guidance | derivation test of the apoptosis by the chemical | medical agent in the Example of this invention.

  The present invention is not limited to the configurations described below, and various modifications are possible within the scope of the claims, and technical means disclosed in different embodiments and examples are used. Embodiments and examples obtained by appropriately combining them are also included in the technical scope of the present invention.

  In addition, when it describes as "A-B" in this specification, the said description shall mean "A or more and B or less."

[1. Cell death promoter)
The cell death promoting agent of the present invention is very safe per se for living organisms, but effectively kills desired cells when used in combination with other drugs or stimuli (specifically, apoptosis) )be able to. An example of an embodiment of the cell death promoter of the present invention will be described below.

  The cell promoting agent of the present embodiment contains a substance that inhibits the binding between cyclin and PP2A B′γ subunit.

  More specifically, the cell death promoting agent of the present embodiment includes a protein containing at least a part of the ELA motif or a nucleic acid containing at least a part of a polynucleotide encoding the protein. It is out.

  “ELA motif (EGF / Erb1-like Auto-phosphorylation motif)” intends a region consisting of 23 amino acids present in the vicinity of the C-terminus of cyclin.

  Examples of the “ELA motif” include a polypeptide corresponding to the first to 23rd amino acids from the amino terminus of the amino acid sequence shown in SEQ ID NO: 1 or 2, and one or several amino acids missing from the polypeptide. Examples include amino acid sequences that have been deleted, substituted, or added.

The cell death promoting agent of the present embodiment is a protein containing at least a part of the polypeptide described in the following (a) or (b) (corresponding to the above-described protein containing at least a part of ELA motif) Or a nucleic acid containing at least a part of a polynucleotide encoding the protein (as described above, a nucleic acid containing at least a part of a polynucleotide encoding a protein containing the ELA motif as at least a part) Corresponding). That means
(A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2;
(B) It consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 or 2, and inhibits the binding between cyclin and PP2A B′γ subunit. Polypeptide.

Further, the cell death promoting agent of the present embodiment contains at least a part of a protein containing at least a part of the polypeptide described in (e) below or a polynucleotide encoding the above protein. A nucleic acid. That means
(E) A polypeptide comprising an amino acid sequence having 90% or more homology with the amino acid sequence shown in SEQ ID NO: 1 or 2, and inhibiting the binding between cyclin and PP2A B′γ subunit.

  In addition, the cell death promoting agent of the present embodiment contains, as at least a part, a protein comprising the polypeptide described in (a), (b) or (e) above, or a polynucleotide encoding the protein. It may contain nucleic acid.

  Here, the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 is a partial peptide of cyclin G1, which is a partial peptide containing an ELA motif (EGF / Erb1-like Auto-phosphorylation motif) of cyclin G1. .

  On the other hand, the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 is a partial peptide of cyclin G2, which is a partial peptide containing an ELA motif (EGF / Erb1-like Auto-phosphorylation motif) of cyclin G2.

  That is, the cell death promoting agent of the present embodiment is a living body (for example, cell, tissue, individual, etc.) containing a protein containing at least a part of the polypeptide described in (a), (b) or (e) described above. As long as it can be administered in any form, any form may be used.

  For example, the cell death promoting agent of the present embodiment may include the protein itself, or a nucleic acid (for example, DNA or RNA) that can generate (for example, translate) the protein in vivo. May be included.

  From the viewpoint of more easily and more reliably killing cells, it can be said that the cell death promoting agent of the present embodiment preferably contains a polynucleotide capable of generating the protein in vivo.

  In the present embodiment, the total number of amino acids constituting the protein containing at least part of the polypeptide described in (a) or (b) is not particularly limited, but may be, for example, 200 or less, 190 Or less, 180 or less, 170 or less, 160 or less, 150 or less, or 140 or less. 130 or less, 120 or less, 110 or less, 100 or less, 90 or less, or 80 or less. Or less, 70 or less, 60 or less, 50 or less, 40 or less, or 30 or less. Good.

  From the viewpoint of ease of administration to a living body (for example, ease of incorporation into cells), protein stability, and elimination of effects other than the desired effect, it can be said that the smaller the total number of amino acids, the better. For example, it is preferably 50 or less, more preferably 40 or less, and most preferably 30 or less.

  In the present specification, in the “amino acid in which one or several amino acids are deleted, substituted or added”, the position where the deletion, substitution or addition occurs is not particularly limited.

  Further, the number of amino acids intended by “one or several amino acids” is not particularly limited, but is preferably 20 amino acids or less, more preferably 19 amino acids or less, and 18 amino acids or less. More preferably, it is more preferably no more than 17 amino acids, still more preferably no more than 16 amino acids, still more preferably no more than 15 amino acids, and no more than 14 amino acids. More preferably, it is 13 amino acids or less, more preferably 12 amino acids or less, further preferably 11 amino acids, more preferably 10 amino acids or less. More preferably, it is 9 amino acids or less, more preferably 8 amino acids or less, and 7 amino acids or less. More preferably, it is 6 amino acids or less, more preferably 5 amino acids or less, further preferably 4 amino acids or less, further preferably 4 amino acids or less. More preferably, it is more preferably 2 amino acids or less, and most preferably 1 amino acid.

  The amino acid substitution is preferably a conservative substitution. The conservative substitution means that a specific amino acid is substituted with another amino acid having the same chemical properties and / or structure as the amino acid. Chemical properties include, for example, hydrophobicity (hydrophobic and hydrophilic), charge (neutral, acidic and basic). Examples of the structure include an aromatic ring, an aliphatic hydrocarbon group, and a carboxyl group that exist as a side chain or a functional group of the side chain.

  Examples of conservative substitutions include, for example, substitution of serine and threonine, substitution of lysine and arginine, and substitution of phenylalanine and tryptophan amino. Of course, the present invention is not limited to these substitutions.

  Moreover, the homology of an amino acid sequence can be calculated | required by a well-known method. Specifically, GENETYX-WIN (manufactured by GENETICS Co., Ltd.) is used according to the GENETYX-WIN manual, for example, a homology search (homology search) between a specific amino acid sequence and an amino acid sequence to be compared is performed, and the same The homology can be calculated as the percentage (%) of amino acids.

  More specifically, the homology can be calculated as the ratio (%) of the number of identical amino acids to the total number of amino acids in the longer amino acid sequence of the amino acid sequences to be compared.

  The polypeptide described in (e) above has a homology of 90% or more with the amino acid sequence shown in SEQ ID NO: 1 or 2, but the homology may be 91% or more. % Or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more It may be 98% or more, or 99% or more.

  The polypeptides described in (a), (b) and (e) described above are polypeptides that inhibit the binding between cyclin and PP2A B′γ subunit.

  Although it does not specifically limit as a kind of PP2A B 'gamma subunit, For example, B'3 subunit, B'2 subunit, or B'1 subunit can be mentioned.

  The method for confirming whether or not the polypeptide is a polypeptide that inhibits the binding between cyclin and PP2A B′γ subunit is not particularly limited. For example, under the conditions in which the polypeptide to be tested exists. This can be confirmed by conducting a coprecipitation test between cyclin and PP2A B′γ subunit.

  For example, immunoprecipitation is performed from a solution in which a cyclin (for example, cyclin G1 or cyclin G2), PP2A B′γ subunit (for example, PP2A B′γ3 subunit), and the polypeptide to be tested coexist. The cyclin is precipitated and separated by a method or the like (the precipitate is referred to as precipitate A). Note that the concentration of the polypeptide to be tested in the solution is not particularly limited. For example, assuming that the concentration of cyclin in the solution and the concentration of PP2A B′γ subunit are substantially the same, The concentration of the polypeptide to be tested is preferably 10 times or more, more preferably 100 times or more the concentration of cyclin.

  As a negative control, from a solution in which cyclin (for example, cyclin G1 or cyclin G2) and PP2A B′γ subunit (for example, PP2A B′γ3 subunit) coexist, immunoprecipitation or the like is used. , Precipitate and separate the cyclin (referred to as precipitate B).

  Next, the PP2A B′γ subunit contained in each of the precipitate A and the precipitate B is quantified. The quantification method is not particularly limited, and examples thereof include a Western blot method using an anti-PP2A B′γ subunit antibody. As the anti-PP2A B′γ3 antibody, a commercially available antibody may be used as appropriate. Of course, it is also possible to use an antibody prepared according to a known method.

  Next, the amount of PP2A B′γ subunit contained in the precipitate A is compared with the amount of PP2A B′γ subunit contained in the precipitate B, and the PP2A B′γ contained in the precipitate A is compared. If the amount of subunit is less than the amount of PP2A B′γ subunit contained in the precipitate B (eg, preferably ½ or less, more preferably 1/5 or less, most preferably 1/10 or less) ), The polypeptide to be tested can be determined to be a polypeptide that inhibits the binding between cyclin and the PP2A B′γ subunit.

  It is also possible to confirm whether the polypeptide is a polypeptide that inhibits the binding between cyclin and the PP2A B′γ subunit by a method different from the above-described method.

  For example, immunoprecipitation is performed from a solution in which a cyclin (for example, cyclin G1 or cyclin G2), PP2A B′γ subunit (for example, PP2A B′γ3 subunit), and the polypeptide to be tested coexist. The PP2A B′γ subunit is precipitated and separated by a method or the like (the precipitate is referred to as precipitate C). Note that the concentration of the polypeptide to be tested in the solution is not particularly limited. For example, assuming that the concentration of cyclin in the solution and the concentration of PP2A B′γ subunit are substantially the same, The concentration of the polypeptide to be tested is preferably 10 times or more, more preferably 100 times or more the concentration of cyclin.

  As a negative control, from a solution in which cyclin (for example, cyclin G1 or cyclin G2) and PP2A B′γ subunit (for example, PP2A B′γ3 subunit) coexist, immunoprecipitation or the like is used. , Precipitate and separate the PP2A B′γ subunit (this precipitate is referred to as precipitate D).

  Next, the cyclins contained in each of the precipitate C and the precipitate D are quantified. The quantification method is not particularly limited, and examples thereof include a Western blot method using an anti-cyclin antibody. A commercially available antibody may be used as appropriate as the anti-cyclin antibody. Of course, it is also possible to use an antibody prepared according to a known method.

  Then, the amount of cyclin contained in the precipitate C is compared with the amount of cyclin contained in the precipitate D, and the amount of cyclin contained in the precipitate C is compared with the amount of cyclin contained in the precipitate D. If it is less than the amount (eg, preferably ½ or less, more preferably 1/5 or less, most preferably 1/10 or less), the polypeptide to be tested is a cyclin and a PP2A B′γ subunit. It can be determined that the polypeptide inhibits binding.

  The cell death promoting agent according to the present embodiment causes the administration target to develop a living organism that has developed DNA damage (for example, DNA double-strand break) or DNA damage (for example, DNA double-strand break). It may be a living body that is subjected to the treatment.

  The cell death promoting agent of the present embodiment is particularly effective in killing a living body that has developed DNA double-strand breaks among damages that occur in DNA (in other words, chromosomes). Therefore, if it is the said structure, a desired biological body can be killed specifically and efficiently.

  In the present specification, “a living body that has developed a DNA double-strand break” is intended to mean a living body in which a DNA double-strand break has already occurred at the time of administration of the cell death promoter of this embodiment. doing.

  For example, the case where the cell death promoting agent of the present embodiment is administered to a living body after an agent that causes DNA double-strand breaks is administered is contemplated. Of course, this case is only an example, and the present embodiment is not limited to this case.

  In addition, in the present specification, “the living body subjected to treatment for causing DNA double-strand break” refers to the time of administration of the cell death promoting agent of the present embodiment, or of the present embodiment. A biological body to which a treatment for causing DNA double strand breakage is performed after administration of a cell death promoting agent is intended.

  For example, when a drug that causes DNA double-strand breaks and the cell death promoting agent of the present embodiment are administered to the living body at the same time, or the living body after the cell death promoting agent of the present embodiment is administered For example, when administering an agent that causes DNA double-strand breaks. Of course, these cases are only examples, and the present embodiment is not limited to these cases.

  Specifically, the living body may be treated with γ-ray irradiation, X-ray irradiation, administration of a topoisomerase I inhibitor (eg, camptothecin administration, irinotecan administration), or administration of a topoisomerase II inhibitor (eg, etoposide administration). It may be a living body that has developed single-strand breaks or a living body that is subjected to treatment for causing DNA double-strand breaks.

  Gamma irradiation, X-ray irradiation, topoisomerase I inhibitor administration (for example, camptothecin administration, irinotecan administration), or topoisomerase II inhibitor administration (for example, etoposide administration) is frequently performed in vivo. Single strand breaks can occur. Therefore, if it is the said structure, a desired biological body can be killed specifically and efficiently.

  The cell death promoting agent of the present embodiment may contain an agent that causes DNA double strand breakage.

  The agent that causes DNA double-strand breaks is not particularly limited, and for example, at least one selected from the group consisting of a topoisomerase I inhibitor (eg, camptothecin, irinotecan) and a topoisomerase II inhibitor (eg, etoposide). There may be.

  The amount of the drug contained in the cell death promoting agent is not particularly limited, but for example, 10 mg or less, 1 mg or less, 0.1 mg or less, 0.01 mg or less, , 0.001 mg or less of the drug can be administered. Of course, the present invention is not limited to the amounts described above.

  The cell death promoting agent of the present embodiment can amplify the effect of the drug. Therefore, the cell death promoting agent of the present embodiment can specifically and efficiently kill a desired living body even if the amount of the contained drug is small.

  As described above, the cell death promoting agent of the present embodiment comprises at least a polynucleotide encoding a protein containing at least a portion of the polypeptide described in (a), (b) or (e) above. Nucleic acids contained as a part may be included. That is, the cell death promoting agent of the present embodiment may contain a nucleic acid (for example, DNA or RNA) that can generate (for example, translate) the protein in vivo.

More specifically, the polynucleotide encoding the protein may be a polynucleotide containing at least part of the following DNA (c) or (d). That means
(C) DNA consisting of the base sequence shown in SEQ ID NO: 3 or 4;
(D) hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the DNA consisting of the base sequence shown in SEQ ID NO: 3 or 4 and binding between cyclin and PP2A B′γ subunit DNA encoding a polypeptide that inhibits.

  Here, the DNA consisting of the nucleotide sequence shown in SEQ ID NO: 3 is a partial peptide of cyclin G1, which encodes a partial peptide containing an ELA motif (EGF / Erb1-like Auto-phosphorylation motif) of cyclin G1. DNA. In other words, it is an example of DNA encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1.

  On the other hand, the DNA consisting of the nucleotide sequence shown in SEQ ID NO: 4 is a partial peptide of cyclin G2, which encodes a partial peptide of cyclin G2 containing an ELA motif (EGF / Erb1-like Auto-phosphorylation motif). DNA. In other words, it is an example of DNA encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2.

  As used herein, the term “stringent conditions” refers to hybridization solutions (50% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7). .6) Incubate overnight at 42 ° C. in 5 × Denhart solution, 10% dextran sulfate, and 20 μg / ml denatured sheared salmon sperm DNA, then in 0.1 × SSC at about 65 ° C. However, depending on the polynucleotide to be hybridized, the washing conditions at high stringency are appropriately changed. For example, when mammalian DNA is used, 0.1% SDS containing 0.1% SDS is used. Washing at 65 ° C. in 5 × SSC (preferably 15 minutes × 2 times) is preferred. When using E. coli-derived DNA, washing at 68 ° C. in 0.1 × SSC containing 0.1% SDS (preferably 15 minutes × 2 times) is preferable, and when using RNA, 0.1% Washing at 68 ° C. in 0.1 × SSC containing SDS (preferably twice for 15 minutes) is preferable. When oligonucleotides are used, in 0.1 × SSC containing 0.1% SDS Washing at the hybridization temperature (preferably 15 minutes × 2 times) is preferred. In addition, the hybridization is described in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2d Ed. , Cold Spring Harbor Laboratory (1989).

  The nucleic acid containing at least a part of the polynucleotide encoding the protein may be one in which the polynucleotide encoding the protein is inserted into an expression vector.

  In this case, the type of the expression vector is not particularly limited. For example, a plasmid, a phage vector, a viral vector (for example, a herpes virus vector, an adenovirus vector, an adeno-associated virus vector, and a retrovirus vector), or a virus particle It can be.

  Of these expression vectors, viral vectors are preferred. This is because the nucleic acid can be stably introduced into the host.

[2. Method for screening active ingredient of cell death promoter]
As described above [1. In the cell death promoting agent], as an example of the present invention, a protein containing at least a part of the polypeptide described in (a), (b) or (e) above, or a polynucleotide encoding the protein A cell death promoter comprising a nucleic acid comprising at least a portion of

  However, the active ingredient of the cell death promoter of the present invention is not limited to the above-mentioned active ingredient, and those screened based on the idea of the present invention can be used as the active ingredient.

  Below, the screening method of the active ingredient contained in a cell death promoter is demonstrated.

[2-1. Screening method (Example 1)]
The screening method of the active ingredient of the cell death promoting agent of the present embodiment,
An administration step of administering a test sample with an agent that causes DNA double-strand breaks at a lower concentration than that used in clinical settings to cultured cells;
A determination step of determining whether or not the cultured cells have undergone apoptosis;
And a determination step of determining that the test sample causing the apoptosis is an active ingredient of a cell death promoting agent.

  In the administration step, mild DNA double-strand breaks are caused to the cultured cells by an agent that causes DNA double-strand breaks at a lower concentration than that used in clinical practice. Normally, such a mild DNA double-strand break does not cause the cultured cells to undergo apoptosis. However, if it can be used as an active ingredient of the cell death promoter of the present invention, the cultured cells undergo apoptosis even with such mild DNA double-strand breaks.

  Although it does not specifically limit as said cultured cell, For example, osteosarcoma cell (for example, U2OS), prostate cancer cell (PC-3, DU-145), tongue cancer cell (SAS), cervical cancer cell (HeLa), pancreas Cancer cells (PANC-1) or ovarian cancer cells (OVCAR-3) can be used.

  The agent that causes DNA double-strand breaks is not particularly limited. For example, at least one selected from the group consisting of a topoisomerase I inhibitor (eg, camptothecin, irinotecan) and a topoisomerase II inhibitor (eg, etoposide). It can be.

  In the present specification, “concentration lower than that used at the clinical site” means, for example, 1/2 or less, 1/3 or less, 1/4 or less, 1/5 or less of the concentration used in the clinical site. 1/6 or less, 1/7 or less, 1/8 or less, 1/9 or less, 1/10 or less, 1/20 or less, 1/30 or less, 1/40 or less, 1/50 or less, 1/60 or less 1/70 or less, 1/80 or less, 1/90 or less, 1/100 or less, 1/150 or less, 1/200 or less, 1/300 or less, 1/400 or less, 1/500 or less, 1/600 or less 1/700 or less, 1/800 or less, 1/900 or less, or 1/1000 or less.

  More specifically, “lower concentration than that used in clinical practice” is 50 μM or less, 40 μM or less, 30 μM or less, 20 μM or less, 10 μM or less, 9 μM or less, 8 μM or less, 7 μM or less, 6 μM or less, 5 μM or less, 4 μM or less, 3 μM or less, 2 μM or less, 1 μM or less, 0.9 μM or less, 0.8 μM or less, 0.7 μM or less, 0.6 μM or less, 0.5 μM or less, 0.4 μM or less, 0.3 μM or less, 0.2 μM Hereinafter, 0.1 μM or less, 0.09 μM or less, 0.08 μM or less, 0.07 μM or less, 0.06 μM or less, 0.05 μM or less, 0.04 μM or less, 0.03 μM or less, 0.02 μM or less, or 0 It may be 0.01 μM or less.

  From the viewpoint of realizing a safer and more sensitive cell death promoter, the lower the concentration of the drug used in the administration step, the better.

  In the determination step, it is determined whether the cultured cell has undergone apoptosis. As a specific method used in the determination step, a known method may be used. For example, FACS (fluorescence activated cell sorting) may be used.

  In the determination step, the test sample that causes apoptosis is determined to be an active ingredient of the cell death promoter.

  Apoptosis may not occur in all of the cultured cells to be observed. For example, 10% or more, preferably 20% or more, more preferably 30% or more, more preferably 40% or more, more preferably 50% or more, more preferably 60% or more, more preferably 70% of the cultured cells to be observed. As described above, if apoptosis is more preferably 80% or more, and most preferably 90% or more, the test sample can be determined to be an active ingredient of a cell death promoter.

  However, the higher the proportion of cultured cells in which apoptosis occurs, the safer and more sensitive cell death promoter can be realized.

[2-2. Screening method (Example 2)]
The screening method of the active ingredient of the cell death promoting agent of the present embodiment,
Irradiating the cultured cells with γ-rays or X-rays weaker than those used in clinical practice, and administering a test sample to the cultured cells;
A determination step of determining whether or not the cultured cells have undergone apoptosis;
And a determination step of determining that the test sample causing the apoptosis is an active ingredient of a cell death promoting agent.

  In the administration step, mild DNA double-strand breaks are caused by irradiating cultured cells with γ-rays or X-rays that are weaker than those used in clinical practice. Normally, such a mild DNA double-strand break does not cause the cultured cells to undergo apoptosis. However, if it can be used as an active ingredient of the cell death promoter of the present invention, the cultured cells undergo apoptosis even with such mild DNA double-strand breaks.

  Although it does not specifically limit as said cultured cell, For example, osteosarcoma cell (for example, U2OS), prostate cancer cell (PC-3, DU-145), tongue cancer cell (SAS), cervical cancer cell (HeLa), pancreas Cancer cells (PANC-1) or ovarian cancer cells (OVCAR-3) can be used.

  The agent that causes DNA double-strand breaks is not particularly limited. For example, at least one selected from the group consisting of a topoisomerase I inhibitor (eg, camptothecin, irinotecan) and a topoisomerase II inhibitor (eg, etoposide). It can be.

  In the present specification, “weaker γ-rays or X-rays than those used at clinical sites” means, for example, ½ or less, １ ／ or less, 1 / 4 or less, 1/5 or less, 1/6 or less, 1/7 or less, 1/8 or less, 1/9 or less, 1/10 or less, 1/20 or less, 1/30 or less, 1/40 or less, 1 / 50 or less, 1/60 or less, 1/70 or less, 1/80 or less, 1/90 or less, 1/100 or less, 1/150 or less, 1/200 or less, 1/300 or less, 1/400 or less, 1 / It may be 500 or less, 1/600 or less, 1/700 or less, 1/800 or less, 1/900 or less, or 1/1000 or less.

  More specifically, “weaker γ-rays or X-rays than those used in clinical settings” are γ-rays or X-rays with an intensity of 100 gray or less, 10 grays or less, 5 grays or less, or 1 gray or less. possible. Of course, the present invention is not limited to γ-rays and X-rays having these intensities.

  From the viewpoint of realizing a safer and more sensitive cell death promoter, it can be said that the lower the intensity of γ rays and X rays used in the administration step, the better.

  The determination process and the determination process of the present embodiment are described in [2-1. The same as the determination step and the determination step in the screening method (Example 1)]. Therefore, the description thereof is omitted here.

[2-3. Screening method (Example 3)]
The screening method of the active ingredient of the cell death promoting agent of the present embodiment,
An administration step of administering an agent that develops a DNA double-strand break in a smaller amount than that used in clinical practice to a nude mouse transplanted with cultured cells, and a test sample;
In the nude mouse, a determination step of determining whether a tumor derived from the transplanted cultured cells is small, and
And a determination step of determining that the test sample that reduces the tumor derived from the transplanted cultured cells is an active ingredient of a cell death promoter.

  In the above administration step, mild DNA double-strand breaks are caused to cultured cells transplanted into nude mice by a drug that causes DNA double-strand breaks at a lower concentration than that used in clinical practice. . Normally, such a mild DNA double-strand break does not cause the cultured cells to undergo apoptosis. However, if it can be used as an active ingredient of the cell death promoter of the present invention, the cultured cells undergo apoptosis even with such mild DNA double-strand breaks. And if a cultured cell raise | generates apoptosis, the tumor originating in the transplanted cultured cell will become small. In addition, if cultured cells undergo apoptosis, the survival rate of nude mice increases.

  Although it does not specifically limit as said cultured cell, For example, osteosarcoma cell (for example, U2OS), prostate cancer cell (PC-3, DU-145), tongue cancer cell (SAS), cervical cancer cell (HeLa), pancreas Cancer cells (PANC-1) or ovarian cancer cells (OVCAR-3) can be used.

  The agent that causes DNA double-strand breaks is not particularly limited. For example, at least one selected from the group consisting of a topoisomerase I inhibitor (eg, camptothecin, irinotecan) and a topoisomerase II inhibitor (eg, etoposide). It can be.

  In the present specification, “small amount than that used in the clinical field” means, for example, 1/2 or less, 1/3 or less, 1/4 or less, 1/5 or less of the concentration used in the clinical field, 1/6 or less, 1/7 or less, 1/8 or less, 1/9 or less, 1/10 or less, 1/20 or less, 1/30 or less, 1/40 or less, 1/50 or less, 1/60 or less, 1/70 or less, 1/80 or less, 1/90 or less, 1/100 or less, 1/150 or less, 1/200 or less, 1/300 or less, 1/400 or less, 1/500 or less, 1/600 or less, It may be 1/700 or less, 1/800 or less, 1/900 or less, or 1/1000 or less.

  More specifically, the “small amount than that used in clinical practice” is 10 mg or less, 1 mg or less, 0.1 mg or less, 0.01 mg or less, or 0.001 mg or less per 100 g body weight of nude mice. May be. Of course, the present invention is not limited to the amounts described above.

  From the viewpoint of realizing a safer and more sensitive cell death promoter, it can be said that the smaller the amount of drug used in the administration step, the better.

  In the determination step, it is determined whether the tumor derived from the cultured cells transplanted into the nude mouse is small. In addition, it does not specifically limit as a specific method used for the said determination process, For example, the diameter of the tumor which arose on the body surface of the nude mouse should just be measured.

  In the determination step, the test sample that reduces the tumor derived from the transplanted cultured cells is determined to be an active ingredient of the cell death promoter.

  For example, in the above determination step, if the diameter of the tumor produced in the nude mouse administered with the test sample is smaller than the diameter of the tumor produced in the nude mouse not administered with the test sample, the test sample is It can be determined to be an active ingredient of a cell death promoter.

  More specifically, when the diameter of a tumor produced in a nude mouse not administered with the test sample is 1, the diameter of the tumor produced in the nude mouse administered with the test sample is ½ or less, preferably 1 / 3 or less, more preferably 1/4 or less, more preferably 1/5 or less, more preferably 1/6 or less, further preferably 1/7 or less, more preferably 1/8 or less, and further preferably 1/9. If it is below or most preferably 1/10 or less, the test sample can be determined to be the active ingredient of the cell death promoter.

  The determination step and the determination step can be different from those described above.

  For example, the determination step may be a step of determining the survival rate of nude mice transplanted with cultured cells.

  Although the specific day which determines a survival rate is not specifically limited, For example, after performing an administration process, 10 days later may be sufficient, 20 days later may be sufficient, and 30 days later may be sufficient. 1 month later, 2 months later, 3 months later, 4 months later, 5 months later, 6 months later, 7 months later, 8 months later, 9 months later, 10 months later, 11 months later, or 1 year later. Of course, the survival rate may be determined after one year.

  When adopting the determination step, the determination step may be a step of determining a test sample administered to a nude mouse having a high survival rate as an active ingredient of a cell death promoter.

  For example, in the above determination step, if the survival rate of the nude mouse administered with the test sample is higher than the survival rate of the nude mouse not administered with the test sample, the test sample is an active ingredient of the cell death promoter. Can be determined.

  More specifically, the survival rate of the nude mouse administered with the test sample is 1.5 times or more, preferably 2 times or more, preferably 3 times or more of the survival rate of the nude mouse not administered with the test sample. Preferably it is 4 times or more, more preferably 5 times or more, more preferably 6 times or more, more preferably 7 times or more, more preferably 8 times or more, more preferably 9 times or more, or most preferably 10 times or more. If so, the test sample can be determined to be the active ingredient of the cell death promoter.

<1. Analysis of binding between Cyclin G1 and PP2A B′γ3 subunit, and binding between Cyclin G2 and PP2A B′γ3 subunit>
<1-1. Description of the polypeptide used in the test>
In this example, a binding domain in Cyclin G1 and a binding domain in PP2A B′γ3 subunit, which are involved in the binding between Cyclin G1 and PP2A B′γ3 subunit, were identified. In this example, a binding domain in Cyclin G2 and a binding domain in PP2A B′γ3 subunit, which are involved in the binding between Cyclin G2 and PP2A B′γ3 subunit, were identified. The test method and test results will be described below.

  For each of Cyclin G1, Cyclin G2, and PP2A B′γ3, various polypeptides used for testing were generated. Outlines of various polypeptides are shown in FIGS. 1 (A) to (C). The full-length amino acid sequences of Cyclin G1, Cyclin G2, and PP2A B′γ3 subunit are shown in SEQ ID NOs: 5, 6, and 7, respectively.

  More specifically, FIG. 1A describes various polypeptides derived from Cyclin G1, and a Flag tag or Myc tag is linked to these polypeptides.

  In FIG. 1A, “F-Cyclin G1” is obtained by linking a Flag tag to a polypeptide corresponding to the full length of Cyclin G1, and “F-G1_N” is the first from the amino terminus of Cyclin G1. A flag tag is linked to a polypeptide corresponding to the 160th amino acid from the amino acid of “F-G1_C”, a polypeptide corresponding to the 295th amino acid from the 154th amino acid from the amino terminus of Cyclin G1 The flag tag is linked to the polypeptide corresponding to the 275th amino acid from the first amino acid from the amino terminus of Cyclin G1, and the “F-G1_ΔELA” is linked to the polypeptide corresponding to the 275th amino acid. “M-G1_ELA + 6” is 2 from the amino terminus of Cyclin G1. To a polypeptide corresponding to the 295 th amino acid from the 7 th amino acid in which Myc tag was connected.

  Although not shown in FIG. 1A, for the test <1-4> described later, a product in which a Myc tag was linked to a polypeptide corresponding to the full length of Cyclin G1 was also prepared.

  FIG. 1B shows various polypeptides derived from Cyclin G2, and a Flag tag or Myc tag is linked to these polypeptides.

  In FIG. 1 (B), “F-Cyclin G2” is obtained by linking a Flag tag to a polypeptide corresponding to the full length of Cyclin G2, and “F-G2_N” is the first from the amino terminus of Cyclin G2. A flag corresponding to a polypeptide corresponding to the 186th amino acid from the amino acid of No. 1, and a flag corresponding to “F-G2_C” is a polypeptide corresponding to the 345th amino acid from the 175th amino acid from the amino terminus of Cyclin G2. The flag tag is linked to the polypeptide corresponding to the 263rd amino acid from the first amino acid from the amino terminus of Cyclin G2, "F-G2_ΔC" F-G2_ΔELA ”is the first amino acid from the amino terminus of Cyclin G2. A flag tag is linked to a polypeptide in which the amino acid at position 264 from amino acid and the amino acid at position 295 to amino acid 345 from the amino terminus are linked, and “M-G2_ELA + 6” is the amino acid of Cyclin G2. A Myc tag is linked to a polypeptide corresponding to the 272nd amino acid from the terminus to the 300th amino acid.

  Although not shown in FIG. 1B, for the test <1-4> described later, a product in which a Myc tag was linked to a polypeptide corresponding to the full length of Cyclin G2 was also prepared.

  FIG. 1 (C) shows various polypeptides derived from PP2A B′γ3, and a Flag tag or Myc tag is linked to these polypeptides.

  In FIG. 1C, “PP2A B′γ3” is obtained by linking a Flag tag or Myc tag to a polypeptide corresponding to the full length of the PP2A B′γ3 subunit, and “B′γ3_1-151” A Flag tag or Myc tag is linked to a polypeptide corresponding to the first to 151st amino acids from the amino terminus of the PP2A B′γ3 subunit, and “B′γ3 — 149-300” is PP2A B A Flag tag or Myc tag is linked to a polypeptide corresponding to the 149th amino acid to the 300th amino acid from the amino terminus of the γ3 subunit, and “B′γ3 — 288-450” is PP2A B′γ3 subunit. Corresponds to amino acids 288 to 450 from the amino terminus of the unit A flag tag or Myc tag is linked to a polypeptide, and “B′γ3 — 451-524” is a polypeptide corresponding to amino acids 451 to 524 from the amino terminus of the PP2A B′γ3 subunit. To which a Flag tag or Myc tag is linked.

<1-2. Determination of binding domain in Cyclin G1 involved in the binding of Cyclin G1 and PP2A B′γ3 subunit>
A solution (20 mM Tris-HCl (pH 7.5)) containing each of the polypeptides shown in FIG. 1 (A) and a Myc tag linked to a polypeptide corresponding to the full length of the PP2A B′γ3 subunit. 200 mM NaCl, 1 mM EDTA, 0.2% Nonidet® P-40, 1 mM dithiothreitol, 1 mM EGTA, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, protease inhibitor cocktail, 20% glycerol) On the other hand, immunoprecipitation treatment (4 ° C., 3 hours) using an anti-Flag tag antibody and protein A-Sepharose 4B (GE healthcare) was performed.

  It was confirmed by Western blotting using an anti-Myc tag antibody whether or not PP2A B′γ3 subunit was contained in the immunoprecipitate. The result is shown in FIG.

  As shown in FIG. 2A, in the case of a solution containing “F-Cyclin G1”, “F-G1_C” and “F-G1_ΔELA”, the PP2A B′γ3 subunit is contained in the immunoprecipitate. It was confirmed that That is, it was confirmed that each of “F-Cyclin G1”, “F-G1_C” and “F-G1_ΔELA” binds to the PP2A B′γ3 subunit. Note that the strength of bonding is shown on the right side of FIG.

<1-3. Determination of binding domain in Cyclin G2 involved in binding of Cyclin G2 to PP2A B′γ3 subunit>
A solution (each described in <1-2>) containing each of the polypeptides shown in FIG. 1B and a Myc tag linked to a polypeptide corresponding to the full length of the PP2A B′γ3 subunit. The same composition) was subjected to immunoprecipitation treatment (4 ° C., 3 hours) using an anti-Flag tag antibody and protein A-Sepharose 4B (GE healthcare).

  It was confirmed by Western blotting using an anti-Myc tag antibody whether or not PP2A B′γ3 subunit was contained in the immunoprecipitate. The result is shown in FIG.

  As shown in FIG. 2 (B), in the case of the solution containing “F-Cyclin G2” and “F-G2_C”, it was confirmed that PP2A B′γ3 subunit was contained in the immunoprecipitate. . That is, it was confirmed that each of “F-Cyclin G2” and “F-G2_C” binds to the PP2A B′γ3 subunit. Note that the strength of the bond is shown on the right side of FIG.

<1-4. Detailed analysis of binding domains in Cyclin G1 or Cyclin G2>
Shorter regions of the binding domain in Cyclin G1 involved in the binding of Cyclin G1 and PP2A B′γ3 subunit and the binding domain in Cyclin G2 involved in the binding of Cyclin G2 and PP2A B′γ3 subunit A test was conducted to narrow down the situation.

  A polypeptide corresponding to the full length of Cyclin G1 and a Myc tag linked to the polypeptide corresponding to the amino acid 267 to 295 from the amino terminus of Cyclin G1 (M-G1_ELA + 6 ), Myc tag linked to a polypeptide corresponding to the full length of Cyclin G2, or Myc tag linked to a polypeptide corresponding to the 272nd amino acid to 300th amino acid from the amino terminus of Cyclin G2 (M-G2_ELA + 6) and a solution having the Flag tag linked to a polypeptide corresponding to the full length of the PP2A B′γ3 subunit (same composition as described in <1-2>) , Anti-Myc tag antibody and protein A-Sepha ose 4B (GE healthcare) Immunoprecipitation treatment with (4 ° C., 3 hours) was performed.

  It was confirmed by Western blotting using an anti-Flag tag antibody whether or not PP2A B′γ3 subunit was contained in the immunoprecipitate. The result is shown in FIG.

  As shown in FIG. 2 (C), in the case of a solution containing “M-Cyclin G1”, “M-G1_ELA + 6”, “M-Cyclin G2” and “M-G2_ELA + 6”, PP2A B ′ It was confirmed that γ3 subunit was contained. That is, it was confirmed that each of “M-Cyclin G1”, “M-G1_ELA + 6”, “M-Cyclin G2” and “M-G2_ELA + 6” binds to the PP2A B′γ3 subunit. Note that the strength of the bond is shown on the right side of FIGS. 1 (A) and 1 (B).

<1-5. Determination of binding domain in PP2A B′γ3 subunit involved in binding between Cyclin G1 and PP2A B′γ3 subunit>
A solution (each having the same composition as described in <1-2>) containing each of the polypeptides shown in FIG. 1C to which the Myc tag is linked and “F-Cyclin G1” in FIG. 1A. On the other hand, immunoprecipitation treatment (4 ° C., 3 hours) using an anti-Myc tag antibody and protein A-Sepharose 4B (GE healthcare) was performed.

  It was confirmed by Western blotting using an anti-Flag tag antibody whether Cyclin G1 was contained in the immunoprecipitate. The result is shown in FIG.

  As shown in FIG. 3A, in the case of a solution containing “M-PP2A B′γ3”, “M-151”, “M-149-300” and “M-451-524”, immunoprecipitation It was confirmed that Cyclin G1 was contained in the product. That is, it was confirmed that each of “M-PP2A B′γ3”, “M-1-151”, “M-149-300” and “M-451-524” binds to Cyclin G1. Note that the strength of the bond is shown on the right side of FIG.

<1-6. Determination of binding domain in PP2A B′γ3 subunit involved in binding of Cyclin G2 to PP2A B′γ3 subunit>
A solution (each described in <1-2>) containing each of the polypeptides shown in FIG. 1C to which a Flag tag is linked, and a polypeptide in which the Myc tag is linked to a polypeptide corresponding to the full length of Cyclin G2. The same composition) was subjected to immunoprecipitation treatment (4 ° C., 3 hours) using an anti-Flag tag antibody and protein A-Sepharose 4B (GE healthcare).

  It was confirmed by Western blotting using an anti-Myc tag antibody whether or not Cyclin G2 was contained in the immunoprecipitate. The result is shown in FIG.

  As shown in FIG. 3B, in the case of a solution containing “F-PP2A B′γ3”, “F-151”, “F-149-300” and “F-451-524”, immunoprecipitation It was confirmed that Cyclin G2 was contained in the product. That is, it was confirmed that each of “F-PP2A B′γ3”, “F-1-151”, “F-149-300” and “F-451-524” binds to Cyclin G2. Note that the strength of the bond is shown on the right side of FIG.

<2. Binding inhibition test>
From the above tests <1-1> to <1-6>, the binding domain in Cyclin G1 involved in the binding between Cyclin G1 and PP2A B′γ3 subunit was determined as “M-G1_ELA + 6 shown in FIG. The binding domain in Cyclin G2 involved in the binding of Cyclin G2 and PP2A B′γ3 subunit is a polypeptide corresponding to “M-G2_ELA + 6” shown in FIG. 1 (B). It became clear that there was.

  Therefore, it was examined whether these polypeptides actually inhibit the binding between Cyclin G1 and PP2A B′γ3 subunit, and the binding between Cyclin G2 and PP2A B′γ3 subunit.

  First, a polypeptide corresponding to “M-G1_ELA + 6” shown in FIG. 1A (referred to as ELAS1 (SEQ ID NO: 1)) and a polypeptide corresponding to “M-G2_ELA + 6” shown in FIG. 1B ( Chemically synthesized ELAS2 (SEQ ID NO: 2)). The specific method of chemical synthesis was in accordance with a well-known method.

<2-1. Binding inhibition effect of ELAS1>
A solution containing GST linked to a polypeptide corresponding to the entire length of GST or Cyclin G1 and a solution linked to a polypeptide corresponding to the full length of PP2A B′γ3 subunit to a Myc tag (<1-2> was prepared.

  The solution was subjected to sedimentation treatment (4 ° C., 3 hours) using Glutathione Sepharose bead (GE healthcare) in the absence of ELAS1 or in the presence of ELAS1.

  It was confirmed by Western blotting using an anti-Myc tag antibody whether or not PP2A B′γ3 subunit was contained in the precipitate. The result is shown in FIG.

  As apparent from FIG. 4 (A), PP2A B′γ3 subunit was contained in the precipitate in the absence of ELAS1, whereas PP2A B ′ was present in the precipitate in the presence of ELAS1. The γ3 subunit was not included.

  That is, it was revealed that the binding between Cyclin G1 and PP2A B′γ3 subunit was inhibited by ELAS1.

<2-2. Binding inhibition effect of ELAS2>
A solution containing GST linked to a polypeptide corresponding to the full length of GST or Cyclin G2 and a solution linked to a polypeptide corresponding to the full length of PP2A B′γ3 to a Myc tag (<1- 2> was prepared.

  The solution was subjected to sedimentation treatment (4 ° C., 3 hours) using Glutathione Sepharose bead (GE healthcare) in the absence of ELAS2 or in the presence of ELAS2.

  It was confirmed by Western blotting using an anti-Myc tag antibody whether or not PP2A B′γ3 subunit was contained in the precipitate. The result is shown in FIG.

  As is clear from FIG. 4B, the precipitate contained PP2A B′γ3 subunit in the absence of ELAS2, whereas PP2A B ′ was present in the precipitate in the presence of ELAS2. The γ3 subunit was not included.

  That is, it was revealed that ELAS2 inhibits the binding between Cyclin G2 and PP2A B′γ3 subunit.

<3. Study on side effects of ELAS1 and ELAS2-1>
From the test <2> described above, it is clear that ELAS1 inhibits the binding between Cyclin G1 and PP2A B′γ3 subunit, and ELAS2 inhibits the binding between Cyclin G2 and PP2A B′γ3 subunit. became. This suggests that ELAS1 and ELAS2 can inhibit the functions of Cyclin G1 and Cyclin G2 in the administered organism.

  Therefore, it was examined whether or not adverse effects can occur in the living body when the functions of Cyclin G1 and Cyclin G2 are inhibited in vivo.

  First, according to a known method, a Cyclin G1 knockout mouse, a Cyclin G2 knockout mouse, and a Cyclin G1 and Cyclin G2 double knockout mouse were prepared. FIG. 5A shows a strategy for producing a Cyclin G1 knockout mouse, and FIG. 5B shows a strategy for producing a Cyclin G2 knockout mouse.

  Cyclin G1 and Cyclin G2 double knockout mice were prepared by mating of Cyclin G1 knockout mice with Cyclin G2 knockout mice.

  Cyclin G1 knockout mice (2 weeks old), Cyclin G2 knockout mice (2 weeks old), and Cyclin G1 and Cyclin G2 double knockout mice (2 weeks old) were each treated with diethylnitrosamine (carcinogen). Diethylnitrosamine (DEN) was administered. Then, 36 weeks after administration, the tumor produced in the liver of each knockout mouse was observed.

  FIG. 6 shows a photograph of a tumor generated in the liver of each knockout mouse. In addition, the circle shown in FIG. 6 has shown the position of the tumor.

  FIG. 7A shows the ratio of the weight of the liver per body weight, and FIG. 7B shows the number of tumors having a diameter of 3 mm or more.

  As is clear from FIGS. 6, 7 (A), and 7 (B), when a carcinogen was administered to wild-type mice, many large tumors were generated in the liver. On the other hand, when the carcinogen was administered to the knockout mouse, the number of tumors produced in the liver was smaller and the size of the tumor was smaller than that of the wild-type mouse.

  The above results indicate that even if the functions of Cyclin G1 and Cyclin G2 in the living body are inhibited by administering ELAS1 and ELAS2, the living body is not adversely affected.

  On the contrary, the above results indicate that if the functions of Cyclin G1 and Cyclin G2 in the living body are inhibited by administering ELAS1 and ELAS2, the living body is hardly carcinogenic.

<4. Test-2 on side effects of ELAS1 and ELAS2>
According to a known method, pTet-On was introduced into U2OS cells, and U2OS cells stably retaining pTet-On were selected in a medium containing G418. Sorted cells were named U2OS / Tet-On cells.

  Next, pTRET3-6Myc into which ELAS1 or ELAS2 was inserted and Linear Hydromycin Marker (Clontech) were introduced into the U2OS / Tet-On cells.

  Next, the expression of ELAS1 or ELAS2 is induced by giving doxycycline (Dox) to U2OS / Tet-On cells into which each gene has been introduced, and the change in the cell cycle of the U2OS / Tet-On cells at this time is observed. did. The change in the cell cycle was analyzed by a well-known method using flow cytometry.

  FIG. 8 (A) shows changes in the expression levels of ELAS1 and ELAS2 in U2OS / Tet-On cells before and after giving doxycycline (Dox). 8A to 8C, “Myc-vec” and “M-vec” indicate U2OS / Tet-On cells into which empty pTRET3-6Myc has been introduced, and “Myc-ELAS1 "And" M-ELAS1 "indicate U2OS / Tet-On cells into which pTRET3-6Myc into which ELAS1 has been inserted has been introduced, and" Myc-ELAS2 "and" M-ELAS2 "refer to pTRET3-in which ELAS2 has been inserted. Shown are U2OS / Tet-On cells into which 6Myc has been introduced.

  As shown in FIG. 8 (A), it was confirmed that expression of ELAS1 or ELAS2 was induced by doxycycline (Dox).

  FIG. 8B and FIG. 8C show the results of flow cytometry. As is clear from FIGS. 8B and 8C, even when ELAS1 and ELAS2 expression was induced in the cells, there was no change in the cell cycle. This indicates that ELAS1 and ELAS2 have very few side effects and are safe for the living body.

<5. Induction of apoptosis by gamma rays>
For the U2OS / Tet-On cells described in <4> above, the effects of ELAS1 and ELAS2 on apoptosis induced by γ rays were examined.

  Specifically, U2OS / Tet-On cells into which pTRAT3-6Myc with ELAS1 was introduced, U2OS / Tet-On cells with ELAS2 inserted, and empty pTRET3-6Myc Each of U2OS / Tet-On cells into which 10 was introduced was irradiated with 10 gray (Gy) of γ-rays, and then the cell cycle (particularly, sub-G1) of each cell was observed over time.

  As shown in FIG. 9A, in the U2OS / Tet-On cells in which ELAS1 and ELAS2 are expressed, the proportion of cells in the sub-G1 phase increased with time. In addition, in the U2OS / Tet-On cells in which ELAS1 is expressed, the change in the proportion of cells in the sub-G1 phase was larger than in U2OS / Tet-On cells in which ELAS2 is expressed. An increase in the proportion of cells in the sub-G1 phase indicates that the cells are dying.

  For example, in U2OS / Tet-On cells in which ELAS1 is expressed, the proportion of cells in the sub-G1 phase is 14.9% 24 hours after irradiation with γ rays, and 48 hours after irradiation with γ rays. The proportion of cells in the sub-G1 phase was 27.2%, and the proportion of cells in the sub-G1 phase was 38.6% 72 hours after irradiation with γ rays.

  Then, in order to confirm whether cell death was due to apoptosis, Annexin V assay and TUNEL assay were performed according to well-known methods.

  FIG. 9B shows the results of Annexin V assay.

  As shown in FIG. 9 (B), in U2OS / Tet-On cells in which ELAS1 is expressed, cells positive for Annexin V increased 72 hours after γ-irradiation. More specifically, cells in “early apoptosis” accounted for approximately 32% of all cells, and cells in “late apoptosis” accounted for approximately 23% of all cells.

  FIG. 9C shows the result of the TUNEL assay. In FIG. 9C, “NT” indicates data before irradiation with γ rays, and “IR72” indicates data at 72 hours after irradiation with γ rays.

  Compared to U2OS / Tet-On cells into which empty pTRET3-6Myc has been introduced, pTRET3-6Myc into which ELAS1 has been inserted and pTRET3-6Myc into which ELAS2 has been inserted In the introduced U2OS / Tet-On cells, cells positive for TUNEL were remarkably increased. In particular, U2OS / Tet-On cells into which pTRET3-6Myc with ELAS1 was introduced were positive for TUNEL compared to U2OS / Tet-On cells with ELAS2 inserted There were a large number of cells.

  The above shows that ELAS1 and ELAS2 can efficiently induce apoptosis by γ rays.

<6. Induction of apoptosis by drugs-1>
When cells are irradiated with gamma rays, the chromosomal DNA of the cells is damaged. There are several types of damage to the chromosome. Examples of the damage include DNA double strand break (DSB) and DNA single strand break (SSB).

  The present inventors conducted the following test based on the hypothesis that apoptosis induced by γ-rays via ELAS1 and ELAS2 depends on the type of damage to the chromosome.

  Among the currently used anti-cancer drugs, various actions such as those that exert anti-cancer action without inducing DSB, and those that exert anti-cancer action while inducing DSB, etc. There are drugs that have a mechanism.

  To date, many drugs are known as drugs that exert anticancer effects while inducing DSB. The induction of DSB can be confirmed by double staining of H2AX and CycG2, which are well known as DSB markers (specifically, if the localization of H2AX and CycG2 match, the cell It can be determined that a DSB has occurred in step 1).

  For example, FIG. 10 shows the results of double staining of H2AX and CycG2 in osteosarcoma (U2OS) cells after treatment with various drugs for 4 hours. Staining was performed according to a well-known method using a commercially available antibody.

  Specifically, for staining H2AX, an anti-γH2AX antibody (manufactured by Millipore) was used as a primary antibody, and an HRP-conjugated anti-rabbit / mouse IgG antibody (manufactured by Capel) was used as a secondary antibody. On the other hand, for staining CycG2, an anti-CycG2 antibody (prepared by a well-known method) was used as the primary antibody, and an HRP-conjugated anti-rabbit / mouse IgG antibody (manufactured by Capel) was used as the secondary antibody.

  Further, as drugs, camptothecin (CPT) (manufactured by sigma), cisplatin (manufactured by LKT Laboratories, Inc.), etoposide (manufactured by sigma), 5-fluorouracil (5-fluorouracil: 5- FU) (manufactured by sigma) and Irinotecan (manufactured by CAMPTO yakult) were used.

  As shown in FIG. 10, osteosarcoma (U2OS) cells were treated with 10 gray γ rays, treated with 10 μM camptothecin (CPT), treated with 20 μg / mL etoposide, 20 μM. When irinotecan was used, the localization of H2AX and CycG2 was consistent within the nucleus. This indicates that gamma radiation, camptothecin, etoposide, and irinotecan cause DSB in osteosarcoma (U2OS) cells.

<7. Induction of apoptosis by drugs-2>
For the U2OS / Tet-On cells described in <4> above, in the presence of various concentrations of camptothecin, irinotecan, cisplatin, or etoposide and doxycycline (Dox), the cell cycle ( In particular, sub-G1) was observed.

  As shown in FIG. 12 (A), the sensitivity of cells to cisplatin that does not cause DSB (in other words, the susceptibility of cell death by cisplatin) was not changed by ELAS1 and ELAS2.

  On the other hand, as shown in FIG. 11A, the sensitivity of cells to camptothecin that causes DSB (in other words, the susceptibility to cell death by camptothecin) was enhanced by ELAS1 and ELAS2.

  In addition, as shown in FIG. 11C, the sensitivity of the cells to irinotecan that causes DSB (in other words, the ease of cell death by irinotecan) was enhanced by ELAS1 and ELAS2.

  In addition, as shown in FIG. 12B, the sensitivity of cells to etoposide causing DSB (in other words, the ease of cell death by etoposide) was enhanced by ELAS1 and ELAS2.

  The present invention can be used as a drug for inducing cell death (for example, cell death due to apoptosis).

  The present invention can be used as a drug for specifically causing cell death (for example, cell death due to apoptosis) in a DNA double-strand break.

  The present invention can be used as an anticancer agent.

Claims (9)

A cell death promoter comprising a protein containing at least a part of an ELA motif, or a nucleic acid containing at least a part of a polynucleotide encoding the protein,
A protein containing at least a part of the polypeptide described in (a) below, or a nucleic acid containing at least a part of a polynucleotide encoding the protein,
The cell death promoting agent, wherein the total number of amino acids constituting the protein containing at least a part of the ELA motif is 50 or less:
(A) polypeptide de comprising an amino acid sequence shown in SEQ ID NO: 1.   The cell death according to claim 1, wherein the administration target is a living body that has developed DNA double-strand breaks, or a living body that is subjected to treatment for causing DNA double-strand breaks. Accelerator.   The living body develops a DNA double-strand break or a DNA double-strand break by γ-ray irradiation, X-ray irradiation, administration of a topoisomerase I inhibitor, or administration of a topoisomerase II inhibitor. 3. The cell death promoter according to claim 2, wherein the cell death promoter is a living body to be treated.   The cell death promoting agent according to any one of claims 1 to 3, further comprising an agent that causes DNA double strand breakage.   The cell death promoter according to claim 4, wherein the drug is at least one selected from the group consisting of a topoisomerase I inhibitor and a topoisomerase II inhibitor. 6. The cell death promoting agent according to claim 1, wherein the polynucleotide encoding the protein is a polynucleotide containing at least a part of the following DNA (c) : Agent:
(C) DNA consisting of the base sequence shown in SEQ ID NO: 3 .   The nucleic acid containing at least a part of the polynucleotide encoding the protein is obtained by inserting the polynucleotide encoding the protein into an expression vector. The cell death promoter according to any one of the above.   The cell death promoting agent according to claim 7, wherein the expression vector is a plasmid, a phage vector, a viral vector, or a viral particle.   The cell death promoter according to any one of claims 1 to 8, which induces apoptosis.