JP2001157585A - Enhancer for anticancer therapy and method for screening the enhancer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for screening a substance which specifically destroys the G2 period checkpoint mechanism of a cancer cell by a DNA- damaging treatment, and a substance which destroys the G2 period checkpoint method mechanism in its cell cycle. SOLUTION: A method for selecting a substance which inhibits the phosphorylation of an oligonucleotide by adding a candidate substance to the phosphorylation system consisting of an oligopeptide having amino acid sequence 1 in the specification, a phosphorylase involved in the G2 period termination, and a labelled phosphorus donor, or a method for selecting a substance which does not allow the DNA amount in a cancer cell to increase after allowing the cell having a defect in the cell cycle G2 period checkpoint mechanism to receive a DNA-damaging treatment, followed by adding a candidate substance to a culture of the cell to incubate, and a substance which destroys the G2 period checkpoint mechanism in the cell cycle are provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a screening method for a cell cycle G2 phase checkpoint mechanism-disrupting substance, a cell cycle G2 phase checkpoint mechanism-disrupting substance selected by the method, and the cell cycle G2.
The present invention relates to an anticancer treatment-enhancing agent comprising a substance that disrupts the early checkpoint mechanism as an active ingredient.

[0002]

2. Description of the Related Art Many anticancer treatments, such as hyperthermia, irradiation with radiation or ultraviolet rays, or administration of anticancer drugs (for example, bleomycin or cisplatin), have DNA damage to cancer cells as a mechanism of action. However, cancer patients often suffer from severe side effects, as these anti-cancer treatments damage both cancer cells and normal cells as well.
In addition, due to this side effect, sufficient anticancer treatment cannot be often performed.

On the other hand, in most normal human cells, when DNA damage occurs, the cell cycle is stopped at the G1 phase checkpoint and the G2 phase checkpoint, and the damaged DNA is repaired. The repair function in cells mainly works during the arrest phase by the G1 checkpoint, and the role of the G2 checkpoint is considered to be small.

[0004] With recent advances in molecular oncology,
The G1 cell cycle checkpoint has been shown to be impaired in most human cancer cells. The majority of cancer cells have p53, Rb,
A tumor suppressor gene such as p16 ^INK4 or p19 ^ARF has a mutation or deletion, or an oncogene such as MDM-2 or cyclin D is overexpressed (References 1 and 2).
In addition to these, overexpression of growth factors and these genes,
Excessive growth signals caused by gain-of-function mutations in those receptors or downstream signaling molecules are thought to be causing cell transformation by rendering the G1 checkpoint dysfunctional.
Exceptionally, some cancer cells have the G2 checkpoint destroyed instead of the G1 checkpoint (Reference 3). This indicates that in the normal cell cycle, the G1 checkpoint is relatively important compared to the G2 checkpoint.
The overaccumulation of mutations is probably fatal to cells,
Mutations that eventually lead to carcinogenesis due to an increase in the mutation rate due to the destruction of the above checkpoint are accumulated in the cells (References 4, 5).

[0005] Interestingly, the G2 phase checkpoint is retained in cancer cells in which the G1 phase checkpoint is impaired. Since normal cells have two independent DNA damage checkpoints, G1 and G2,
If the G2 phase checkpoint is selectively destroyed by any treatment, cancer cells whose G1 phase checkpoint has already been destroyed are more susceptible to DNA damage treatment than normal cells with an intact G1 phase checkpoint. It is expected to become. For example, it has been reported that non-specific G2 checkpoint disruption by caffeine or the like is effective in sensitizing p53-deficient cancer cells to DNA damage (references 6-7).

According to the current hypothesis regarding the G2 phase checkpoint, DNA damage activates protein kinases Chk1 and HuCds1 / Chk2 by activating rad3 ⁺ / ATM (refs. 11-14). Then Chk1 and Huk
Cds1 / Chk2 phosphorylates serine 216 of Cdc25C in humans and promotes binding to 14-3-3 (Ref. 15-).
17). In fission yeast, Cdc25C is sequestered in the cytoplasm by binding of 14-3-3 and Cdc25C (ref. 18), and as a result, the activation of Cdc2 / cyclin B by Cdc25C is inhibited. Is induced, and the DNA is repaired (references 19 to 21).

[0007]

As described above, in cancer cells in which the G1 phase checkpoint is impaired,
Furthermore, if the G2 phase checkpoint is selectively destroyed, it becomes susceptible to DNA damage treatment such as an anticancer agent,
It is expected that effective cancer treatment will be possible. Also,
In such treatment, cancer cells that do not have the two checkpoint mechanisms G1 and G2 are targeted, so that cancer cells can be killed even with a relatively small amount of anticancer drug, It is possible to greatly reduce side effects on cells.

[0008] This application was made in view of the above circumstances, and a method for screening a novel substance capable of selectively destroying the G2 checkpoint mechanism of the cell cycle, It is an object of the present invention to provide a selected substance that disrupts the checkpoint mechanism of the cell cycle G2 phase.

Another object of the present invention is to provide an anticancer treatment enhancer comprising the cell cycle G2 phase checkpoint mechanism disrupting substance as an active ingredient.

[0010]

This application provides the following inventions (1) to (13) to solve the above-mentioned problems. (1) A candidate substance is added to a phosphorylation reaction system comprising an oligopeptide having the amino acid sequence of SEQ ID NO: 1, a kinase involved in G2 arrest, and a labeled phosphorus donor to inhibit phosphorylation of the oligonucleotide. A method for screening a substance that disrupts the cell cycle G2 phase checkpoint mechanism in anticancer therapy, which comprises selecting a substance to be treated. (2) The kinase is hChk1, HuCds1 / Chk2, or a fusion protein of these enzymes with other proteins, or DN.
The screening method according to the invention (1), which is an extract of A-damaged cells. (3) 2nd and 3rd position Xaa in SEQ ID NO: 1 is Gl
y, Leu, Ser or Arg, and Xaa at positions 6 to 8 are Gly, Le
The screening method according to the above invention (1), which is u, Ser, Met, Pro or Glu. (4) Xaa at position 2 in SEQ ID NO: 1 is Gly, Leu, Ser or Arg, Xaa at position 3 is Gly, Leu or Ser, and Xaa at position 6 is
Gly, Met, Pro or Glu, 7th position Xaa is Gly, Leu or P
ro, the screening method according to the above invention (3), wherein Xaa at position 8 is Gly, Met, Ser or Glu. (5) The second position Xaa in SEQ ID NO: 1 is Arg, and the third position Xaa is
Ser, 6th position Xaa is Met, 7th position Xaa is Pro, 8th position Xaa is Gl
The screening method according to the invention (4), which is u. (6) the above-mentioned invention, wherein the phosphorylating enzyme is hChk1, HuCds1 / Chk2, or a fusion protein of these enzymes and another protein;
The screening method according to any one of (3) to (5). (7) A cell having a defect in the cell cycle G1 phase checkpoint mechanism is subjected to DNA damaging treatment, a candidate substance is added to the culture medium of the cell, and the cells are cultured. A method for screening a substance that disrupts the cell cycle G2 phase checkpoint mechanism, wherein the substance does not increase the number of cells. (8) G2 / M in cells treated with DNA damage
G2 / M by drug treatment that does not increase the cell population with DNA amounts corresponding to the
(7) The screening method according to the above-mentioned invention (7), wherein a substance which increases the cell population having a DNA amount corresponding to the phase is selected. (9) A substance that disrupts the cell cycle G2 phase checkpoint mechanism selected by the method according to any one of the inventions (1) to (8). (10) The substance that disrupts the cell cycle G2 phase checkpoint mechanism of the invention (9), which is a compound having a main chain composed of carbon, oxygen and nitrogen, and tyrosine side chains, proline side chains and asparagine side chains. (11) The substance disrupting the cell cycle G2 phase checkpoint mechanism according to the invention (9), which is an oligopeptide having the amino acid sequence of SEQ ID NO: 2. (12) 2nd and 3rd position Xaa in SEQ ID NO: 2 is Gl
y, Leu, Ser or Arg, wherein Xaa at positions 5 to 8 are Ser, Gl
the cell cycle G of the invention (11), which is y, Met, Pro or Glu;
Second-phase checkpoint mechanism destructive substance. (13) The cell cycle G2 according to any one of the inventions (9) to (12).
An anti-cancer treatment enhancer comprising a checkpoint mechanism disrupting substance or a derivative thereof as an active ingredient.

Hereinafter, embodiments of the present invention will be described in detail.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The screening method of the invention (1) of this application can be carried out, for example, as follows.

[0013] A candidate substance is added to an in vitro phosphorylation reaction system comprising a substrate (oligopeptide having the amino acid sequence of SEQ ID NO: 1), a phosphorylating enzyme involved in G2 arrest, and a labeled phosphorus donor. Select a substance that inhibits oxidation. The substance selected in this manner is a substance that inhibits phosphorylation of a substrate by inhibiting the activity of a kinase, and thus can be specified as a substance that disrupts the cell cycle G2 phase checkpoint mechanism.

As the labeled phosphorus donor, for example, γ labeled with the radioactive isotope [32] P as shown in the Examples
-ATP or the like can be used. Further, as the phosphorylase involved in the G2 arrest of the cell, the full length of the human-derived phosphorylase hChk1 or HuCds1 / Chk2 can be used. In addition, these enzymes and other proteins (eg, G
ST, MBP, etc.). Furthermore, an extract of cells stimulated by DNA damaging treatment such as an anticancer agent can also be used (see the above description of the invention).
(2)).

[0015] Oligopeptides as substrates include, specifically, Xaa at positions 2 and 3 in SEQ ID NO: 1 being Gly;
Leu, Ser or Arg, wherein Xaa at positions 6-8 are Gly, Leu,
Oligopeptides that are Ser, Met, Pro or Glu
(3)). More specifically, Xaa at positions 2 and 3 in SEQ ID NO: 1 is Gly, Leu, Ser or Arg, and Xaa at positions 6 to 8 are Gly, Leu, Ser, Met, Pro or Gl.
u is an oligopeptide (invention (4)), and most specifically, the second position Xaa in SEQ ID NO: 1 is Arg and the third position Xaa
Is Ser, 6th position Xaa is Met, 7th position Xaa is Pro, and 8th position Xaa is
An oligopeptide that is Glu (Invention (5): SEQ ID NO: 3).

The screening method of the invention (7) of this application can be carried out, for example, as follows.
First, DNA damage treatment is performed on cells having a defect in the cell cycle G1 phase checkpoint mechanism, such as the human leukemia cell line Jurkat. Examples of the DNA damaging treatment include a treatment in which an anticancer agent such as bleomycin is added to a cell culture medium, or a treatment in which cells are irradiated with radiation. Then, a candidate substance is added to the culture medium of the cells, and after culturing for about 10 to 48 hours, the DNA amount of the cells is measured. For example, the culture supernatant can be discarded, the cells can be suspended in a surfactant solution such as propidium iodide and NP-40, and the DNA amount of the cells can be measured using a flow cytometer. This amount of DNA corresponds to G2 of the control cells.
If the increase is equal to the / M phase, it is determined that the candidate drug added to the medium has not destroyed the G2 phase checkpoint mechanism. On the other hand, a substance that does not increase the amount of DNA at the same level as the G2 / M phase of control cells is identified as a G2 phase checkpoint mechanism disrupting substance.

Further, in the screening method of the invention (8) of the present application, the amount of DNA of the cell selected by the method of the invention (7) is reduced by the treatment with a drug which causes cell cycle M phase arrest. Select substances that increase. That is, the cells are treated with an agent that causes M-phase arrest, such as colchicine, to stop the cell cycle at M-phase, and the substance selected in the above-mentioned invention (7) is added to the culture medium of the cells. At this time, a substance that increases the amount of DNA equivalent to that in the G2 / M phase does not affect the arrest in the M phase, and is specified as a substance that selectively destroys only the G2 / phase checkpoint mechanism.

The invention (9) of the present application is a cell cycle G2 phase checkpoint mechanism disrupting substance selected by the screening method of the inventions (1) to (8) as described above. Examples of such a substance include a compound having a main chain composed of carbon, oxygen and nitrogen, and a tyrosine side chain and a proline side chain (Invention (10)). For example, FIG.
B is a compound whose structure is exemplified.

Furthermore, an oligopeptide having the amino acid sequence of SEQ ID NO: 2 (Invention (11)) can also be exemplified as the substance that disrupts the checkpoint mechanism in the G2 phase of the cell cycle. Such an oligopeptide is, for example, SEQ ID NO: 2
2nd and 3rd position Xaa in Gly, Leu, Ser or
Arg, 5th to 8th position Xaa is Ser, Gly, Met, Pro or
An oligopeptide that is Glu (Invention (12)), specifically, a synthetic peptide whose structure is shown in FIG. 1A. Such an oligopeptide can be prepared by a known solid phase peptide synthesis method or the like.

The invention (13) is an anticancer treatment enhancer comprising the cell cycle G2 phase checkpoint mechanism disrupting substance according to the invention (9) to (12) as an active ingredient. This drug can be administered to a cancer tissue to destroy the G2 phase checkpoint of the cancer cells, and at the same time, can be used in a cancer treatment method of killing the cancer cells by performing anticancer treatment such as administration of an anticancer agent. That is, the G2 phase checkpoint mechanism-disrupting substance is G
Phosphorylases hChk1 and HuCds1 / involved in the two-phase arrest
In order to suppress Chk2 activity, cancer cells that have already lost the G1 checkpoint function are arrested in G2
NA repair cannot be performed, and treatment with an anticancer agent or the like can be made to act more effectively. This enhancer, for example,
The active ingredient G2 phase checkpoint mechanism-disrupting substance can be formulated by dissolving or dispersing in a suitable solution carrier, or mixing or adsorbing with a suitable powder carrier.

Hereinafter, the invention of this application will be described in more detail and specifically with reference to examples, but the invention of this application is not limited by the following examples.

[0022]

EXAMPLES Example 1 Oligopeptides YPN (SEQ ID NO: 4), AAA (SEQ ID NO: 5) and 4aa (SEQ ID NO: 6) were prepared by a known solid-phase peptide synthesis method.

8 × 10^FiveIndividual Jurkat cells, 10% fetal bovine serum
RPMI 1640 medium at 37 ° C, 5% CO _TwoAnd cultured. this
Add 10 μg / ml bleomycin and 20 μg / ml to the medium.
Of oligopeptide YPN, AAA or 4aa of
After 0, 6, 12, and 24 hours, the amount of DNA was quantified. DNA
Quantitation is in buffer containing propidium iodide and NP-40
The cells were suspended in the sample, and the measurement was performed using a flow cytometer.

The results are as shown in FIG. The left side of FIG. 2 shows the measurement results of the flow cytometer, and the right side is a graph showing the ratio of cells for each DNA amount. As is clear from the results of FIG. 2, the oligopeptide YPN
Cells added to the medium did not increase the amount of DNA in the G2 / M phase.

Further, the amount of DNA in each cell cycle was similarly quantified using colchicine instead of pleomycin.
The results are as shown in FIG. 3, and the oligopeptide YPN, whose DNA amount did not increase by bleomycin treatment, was obtained.
Also, as with other oligopeptides, colchicine treatment gives D
The amount of NA was increased.

From the above results, the oligopeptide YPN
Was confirmed to be a substance that specifically disrupts the cell cycle G2 phase checkpoint mechanism. Example 2 A synthetic peptide consisting of the amino acid sequence of SEQ ID NO: 7 was used as a substrate, recombinant hChk1 and HuCds1 / Chk2 were used as a phosphorylating enzyme, and a phosphorylation reaction was performed using γ-ATP labeled with a radioactive isotope [32] P. , Oligopeptide TAT-S21
6A (SEQ ID NO: 8) was added to detect reaction inhibition. The reaction was carried out at 30 ° C. for 15 minutes, after which each peptide was separated by 15% SDS-PAGE, and the degree of reaction inhibition was detected by autoradiography.

The results are as shown in FIG.
-S216A inhibited the phosphorylation of the synthetic peptide (SEQ ID NO: 7) by recombinant hChk1 and HuCds1 / Chk2. From the above results, oligopeptide TAT-S216A
Has been confirmed to be a substance that specifically disrupts the cell cycle G2 phase checkpoint mechanism.

Incidentally, TAT-S216A is one of the prior inventions (Japanese Patent Application No. 11-269398) by the inventors of the present application. Example 3 Various amino acid sequences shown in Table 1 were linked to a TAT sequence (N-terminal 11 amino acid sequences of SEQ ID NOs: 4-6, 8, and 9) to synthesize oligopeptides, and G2 in cultured cells
Of the initial checkpoint mechanism (same method as in Example 1), the inhibitory effect of hChk1 and HuCds1 / Chk2 on the phosphorylation reaction of the substrate peptide (SEQ ID NO: 7), and the degree of phosphorylation of each oligopeptide.

The results are as shown in Table 1.

[0030]

[Table 1]

Example 3 Human gastric cancer cell line CO-4 was inoculated subcutaneously into nude mice.
After confirming the engraftment, the anticancer drug CDDP (1.5 mg / kg) or 5-FU (20 mg / kg) was intraperitoneally administered, and the oligopeptide TAT-S216 (SEQ ID NO: 9) 20 mg / kg was added at 0.5 mg / kg.
μl was injected locally into the tumor three or five times a week, the tumor was excised and weighed.

The results are shown in FIG. 5. When the oligopeptide TAT-S216 (Japanese Patent Application No. 11-269398) was administered three times a week, the tumor weight was significantly reduced, and the anticancer agent CD
It was confirmed that the effects of DP (upper part in FIG. 5) and 5-FU (lower part in FIG. 5) were enhanced.

[0033]

As described above in detail, according to the invention of the present application, it is possible to screen a substance capable of specifically destroying the G2 phase checkpoint mechanism of cancer cells by DNA damage treatment and to reliably screen this substance. A possible method is provided. The treatment of the G2 phase checkpoint mechanism-disrupting substance makes the cancer cells sensitive to anticancer treatment such as an anticancer agent, so that cancer treatment can be performed more effectively.

[0034]

[Sequence List] <110> Japan Science and Technology Corporation <120> Enhancer of anti-cancer therapy and screening method thereof <130> NP99464-YS <160> 9 <210> 1 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthesized opeptide <400> 1 Tyr Xaa Xaa Pro Ser Xaa Xaa Xaa Asn 1 5 <210> 2 <211> 9 <212> PRT <213> Artificial Sequence < 220> <223> Synthesized peptide <400> 2 Tyr Xaa Xaa Pro Xaa Xaa Xaa Xaa Asn 1 5 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthesized peptide <400> 3 Tyr Arg Ser Pro Ser Met Pro Glu Asn 1 5 <210> 4 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthesized peptide <400> 4 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Tyr Gly Gly Pro Gly 1 5 10 15 Gly Gly Gly Asn 20 <210> 5 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Synthesized peptide <400> 5 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Leu Ala Arg Ser Ala 1 5 10 15 Ser Met Pro Glu Ala Leu 20 <210> 6 <211> 1 8 <212> PRT <213> Artificial Sequence <220> <223> Synthesized peptide <400> 6 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Gly Ser Pro Ala 1 5 10 15 Met Pro <210> 7 <211 > 11 <212> PRT <213> Artificial Sequence <220> <223> Synthesized peptide <400> 7 Leu Tyr Arg Ser Pro Ser Met Pro Glu Asn Leu 1 5 10 <210> 8 <211> 22 <212> PRT < 213> Artificial Sequence <220> <223> Synthesized peptide <400> 8 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Leu Tyr Arg Ser Pro 1 5 10 15 Ala Met Pro Glu Ala Leu 20 <210> 9 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Synthesized peptide <400> 9 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Leu Tyr Arg Ser Pro 1 5 10 15 Ser Met Pro Glu Ala Leu 20

[0035]

[References]

1. Levine, AJ p53, the cellular gatekeeper
for growth and division. Cell 88, 323-331 (1997). 2. Larsen, CJ Contribution of the dual cod
ing capacity of the p16INK4a / MTS1 / CDKN2 locus to h
uman malignancies. Prog. Cell. Cycle. Res 3, 109-1
24 (1997). 3. Kawabe, T., Muslin, AJ & Korsmeyer, S.
J. Hox11 interacts with protein phosphatases PP2A a
nd PP1 and disrupts a G2 / M cell-cycle checkpoint.
Nature 385, 454-458 (1997). 4. Hartwell, LH & Kastan, MB Cell cycle
control and cancer. Science 266, 1821-1828 (199
Four). 5. Paulovich, AG, Toczyski, DP & Hartwel
l, LH When checkpoints fail. Cell 88, 315-321 (1
997). 6. Rowley, R. Reduction of radiation-induced
G2 arrest by caffeine. Rad. Res 129, 224-227 (199
2). 7. Yao, SL et al. Selective radiosensitiza
tion of p53-deficientcells by caffeine-mediated ac
tivation of p34 ^cdc2 kinase.Nature Med2, 1140-1143
(1996). 8. DeFrank, JS, Tang, W. & Powell, SN p5
3-null cells are moresensitive to ultraviolet ligh
t only in the presence of caffeine.Cancer Res 56,
5365-5368 (1996). 9. Wang, SW, Norbury, C., Harris, AL, To
da, T. Caffeine can override the SM checkpoint in
fission yeast. J. Cell Sci. 112, 927-937 (1999). Ten. Wang, Q., Fan, S, Eastman, A., Worland,
RJ, Sausville, EA & OiConnor, RM UCN-01: a po
tent abrogator of G2 checkpoint functionin cancer
cells with disrupted p53. J. Natl. Cancer. Inst 8
8, 956-965 (1996). 11． Walworth, N., Davey, S., Beach, D. Fissio
n yeast chkl protein kinase links the Rad checkpoi
nt pathway to cdc 2. Nature 363, 368-371 (1993). 12． Walworth, NC, Bernards, R. rad-dependen
t-response of the chkl-encoded protein kinase at t
he DNA damage checkpoint. Science 271,353-356 (199
6). 13. Matsuoka, S., Huang, M., Ellcdgc, SJ Li
nkage of ATM to cell cycle regulation by the Chk2
protein kinase. Science 282, 1893-1897 (1998). 14. Zeng, Y. et al. Replication checkpoint re
quires phosphorylationof the phosphatase Cdc25 by
Cds1 or Chk1. Nature 395, 507-510 (1998). 15． Furnari, B., Rhind, N., Russell, P. Cdc25C
mitotic inducer tergeted by chk1 DNA damage check
point kinase. Science 277, 1495-1497 (1997). 16． Sanchez, Y. et al. Conservation of the ch
k1 checkpoint pathway in mammals: linkage of DNA da
mage to cdk regulation through cdc25.Science 277,
1497-1501 (1997). 17． Peng, CY et al. Mitotic and G2 checkpoi
nt control: regulationof 14-3-3 protein binding by
phosphorylation of cdc25C on serine-216. Science
277, 1501-1505 (1997). 18． Lopez-Girona, A., Furnari, B., Mondesert,
O., Russel, P. Nuclear localization of Cdc25 is r
egulated by DNA damage and a 14-3-3 protein.Natur
e 397, 172-175 (1999). 19. Nurse, P. Checkpoint pathways come of ag
e. Cell 91, 865-867 (1997). 20. Weinert, TA DNA damage checkpoint meets
the cell cycle engine. Science 277, 1450-1451 (19
97). twenty one. Russell, P. Checkpoints on the road to mi
tosis. TIBS 23, 399-402 (1998).

[Brief description of the drawings]

FIG. 1 shows the structures of the oligopeptide (A) of the present invention and the compound (B).

FIG. 2 is a flow cytometer measurement result (left) relating to the DNA amount of Jurkat cells cultured by adding the oligopeptide of the present invention and bleomycin to a medium, and a graph (right) showing the ratio of cells for each DNA amount. is there.

3 is a flow cytometer measurement result in which the amount of DNA in each cell cycle was similarly quantified using colchicine instead of pleomycin in the measurement of FIG.

FIG. 4. Chk1 and Chk2 / HcCds1 of TAT-S216A.
It is a blotting figure which shows the experiment result of activity inhibition. 4 times the amount of TAT-S2 compared to the synthetic peptide (10 μM) used as a substrate
At 16 A (40 μM), the activities of Chk1 and Chk2 / HcCds1 were inhibited.

FIG. 5: Nude mice inoculated subcutaneously with human gastric cancer cell line CO-4 were intraperitoneally administered with anti-cancer drug CDDP or 5-FU, and oligopeptide TAT-S216 was administered three times a week or 5 times.
It is a graph which shows the tumor weight at the time of local injection to a tumor.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI Theme coat ゛ (Reference) A61P 43/00 121 C12N 9/12 4C084 C07K 7/00 C12P 21/02 C 4H045 C12N 9/12 C12Q 1 / 48 Z C12P 21/02 1/68 Z C12Q 1/48 G01N 33/15 Z 1/68 33/50 Z G01N 33/15 C12N 15/00 ZNAA 33/50 A61K 37/02 F term (reference) 2G045 AA26 AA34 AA40 BB20 CB02 DA13 DA36 DA78 FB01 FB07 4B024 AA01 AA11 BA10 DA02 GA30 HA20 4B050 CC03 DD11 LL01 LL03 LL05 4B063 QA05 QQ21 QQ41 QQ61 QQ79 QR07 QR42 QR48 QR57 QR77 QS39 QX02 QX074B01A01 CC01 A0201 ZB262 4H045 AA10 AA20 AA30 BA15 EA28 EA50 FA33

Claims (13)

[Claims] Claims: 1. A candidate substance is added to a phosphorylation reaction system comprising an oligopeptide having the amino acid sequence of SEQ ID NO: 1, a kinase involved in G2 arrest, and a labeled phosphorus donor, and phosphorylation of the oligopeptide A method for screening a substance that disrupts the checkpoint mechanism of the cell cycle G2 phase of anticancer therapy, which comprises selecting a substance that inhibits a cell cycle. 2. The phosphorylating enzyme is hChk1, HuCds1 / Chk.
2. The screening method according to claim 1, which is a fusion protein of these enzymes and another protein, or an extract of DNA-damaged cells. 3. Positions 2 and 3 in SEQ ID NO: 1.
Xaa is Gly, Leu, Ser or Arg, and Xaa at the 6th to 8th positions is
The screening method according to claim 1, wherein the screening method is Gly, Leu, Ser, Met, Pro or Glu. 4. Xaa at position 2 in SEQ ID NO: 1 is Gly, Le
u, Ser or Arg, position 3 Xaa is Gly, Leu or Ser, position 6 Xaa is Gly, Met, Pro or Glu, position 7 Xaa is Gly, L
4. The screening method according to claim 3, wherein eu or Pro, and Xaa at position 8 is Gly, Met, Ser or Glu. 5. The Xaa at position 2 in SEQ ID NO: 1 is Arg, the Xaa at position 3 is Ser, the Xaa at position 6 is Met, the Xaa at position 7 is Pro, and the amino acid at position 8 is Pro.
The screening method according to claim 4, wherein the position Xaa is Glu. 6. The phosphorylating enzyme is hChk1, HuCds1 / Chk.
6. The screening method according to any one of claims 3 to 5, which is a fusion protein of the enzyme and another protein. 7. A cell having a defect in the G1 phase checkpoint mechanism of the cell cycle is subjected to DNA damaging treatment, a candidate substance is added to a culture medium of the cell, and the cells are cultured. A method for screening a substance that disrupts the cell cycle G2 phase checkpoint mechanism, comprising selecting a substance that does not increase the cell population. 8. A G2 / M phase by a drug treatment that does not increase the cell population of the DNA amount corresponding to the G2 / M phase in the cells that have been subjected to the DNA damaging treatment, and that causes cell cycle M phase arrest. The screening method according to claim 7, wherein a substance that increases the cell population of the DNA amount is selected. 9. A G2 phase checkpoint mechanism disrupting substance selected by the method of claim 1. 10. The G2 checkpoint mechanism disrupting substance according to claim 9, which is a compound having a main chain consisting of carbon, oxygen and nitrogen, and tyrosine side chains, proline side chains and asparagine side chains. 11. The substance that disrupts the cell cycle G2 phase checkpoint mechanism according to claim 9, which is an oligopeptide having the amino acid sequence of SEQ ID NO: 2. 12. Positions 2 and 3 in SEQ ID NO: 2
Position Xaa is Gly, Leu, Ser or Arg, and the 5th to 8th position Xaa
Is Ser, Gly, Met, Pro or Glu. 13. An anticancer treatment-enhancing agent comprising the cell cycle G2 phase checkpoint mechanism disrupting substance or its derivative according to any one of claims 9 to 12 as an active ingredient.