JP2003038200A - Method for testing sensitivity of tumor cell to anticancer agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide acting markers for antitumor effect of E7070 and its related compound when E7070 or the related compound is allowed to act on tumor cells. SOLUTION: The sensitivity of tumor cells to an anticancer agent (E7070 or its related compound) is carried out as follows: tumor cells are taken out from a cancer patient to whom the anticancer agent has been administered, and the expression levels of genes described in Table 3 and Table 4 are determined; or the anticancer agent is allowed to act on the tumor cells taken out from a cancer patient, and the expression levels of the genes described in Table 3 and Table 4 are determined; and in the case where the expression level of the gene described in Table 3 is increased, or that of the gene described in Table 4 is decreased, it is judged that the tumor cells are sensitive to the anticancer agent.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for assaying whether tumor cells are sensitive to an anticancer drug.

[0002]

2. Description of the Related Art In conventional clinical trials of anti-cancer agents, first, a toxicity profile and maximum recommended dose are determined in a phase I trial, and then a response rate using the tumor shrinkage rate as a criterion for efficacy in a phase II trial ( Response rate has been evaluated as a drug. On the other hand, with the recent progress in cancer biology, drugs with a new action mechanism that inhibits intracellular signal transduction system and angiogenesis are under active research and development. It is conceivable that it is not always necessary to administer the maximum recommended dose close to the toxic dose for these novel anticancer agents. Furthermore, it is presumed that the effect of the drug can be judged more appropriately by using, as an index, improvement of QOL (Quality of Life) associated with suppression of tumor growth or prolongation of life rather than tumor shrinkage. In this case, in order to confirm the effect of the drug more logically and specifically, the changes in biological markers closely related to the tumor growth suppression mechanism were surrogateed.
It is desired to use it as a marker.

Generally, in anticancer therapy, the reactivity of an organism upon administration of an anticancer drug largely depends on the sensitivity of the tumor cells targeted by the drug to the drug. The susceptibility of tumor cells to drugs varies greatly depending on the tumor cells. Such a difference in sensitivity is due to a quantitative or qualitative difference in the target molecule of the drug or a factor related thereto or the acquisition of drug resistance. Based on such a background, it is possible to measure the change of tumor cells specifically caused when the target tumor cells show sensitivity to a drug, using a tumor tissue obtained by biopsy or the like. If possible, this can be used as a surrogate marker for early determination of drug effects, establishment of treatment methods, selection of new treatment methods, and the like, which is extremely useful. Further, from the tumor tissue obtained by biopsy or the like prior to the treatment, drug treatment is performed after separating the tumor cells according to a conventional method, and whether or not the tumor cells are drug-sensitive is measured by the change of the surrogate marker. Therefore, it becomes possible to predict in advance whether or not the treatment with the drug will be effective, which is extremely useful clinically. It is important that the change of the surrogate marker is specific to the antitumor effect, and that it can be measured with high sensitivity. Specifically, quantification of gene expression fluctuations specific to the antitumor effect of drugs, analysis of protein quantitative fluctuations due to changes in those gene expressions, and further analysis of functional changes accompanying such changes, etc. Can be a marker.

E7070 (N- (3-chloro-7-indolyl) -1,4-benz
enedisulfonamide) is a compound with an antitumor effect that targets the G1 phase of the cell cycle and is currently in clinical development (Takashi Owa, Hiroshi Yoshino, Tatsuo Okauch).
i, Kentaro Yoshimatsu, Yoichi Ozawa, Naoko Hata Su
gi, Takeshi Nagasu, Nozomu Koyanagi and KyosukeKi
toh, J. Med. Chem, 1999, 42, 3789-3799).

The spectrum of the cytostatic activity of this compound on various tumor cells is different from any of the existing anticancer agents, and its effect is expected as an anticancer agent having a new mechanism of action. In order to rapidly advance the clinical development of this drug and establish a clinical treatment method at an early stage, the treatment is efficiently advanced based on the established treatment method, and the patient's QO
In order to contribute to the improvement of L, it is desirable to discover and apply a surrogate marker that can be used specifically during the administration of this drug.

In recent years, various DNA microarrays have been used,
A method for simultaneously detecting the expression level of many genes has been established and applied to a wide range of purposes (Schena M, Shalon
D, Davis RW, Brown PO, Science 1995, 270, 467-70,
Lockhart, DJ, Dong, H., Byrne, MC, Folletti
e, MT, Gallo, MV, Chee, MS, Mittmann, M., Wa
ngC., Kobayashi, M., Horton, H. Brown, EL, Natur
e Biotechnology, 1996,14, 1675-1680).

In the field of cancer research as well, research using this DNA microarray has been actively conducted. DNA for example
In a study that analyzed diffuse large B-cell lymphoma (DLBCL) by expression analysis using a microarray, DLBCL was classified into two different types according to the difference in gene expression profile, and this classification was prognostic. Has been shown to be linked to the prediction of (Alizadeh
AA, Eisen MB, DavisRE, Ma C, Lossos IS, Rosenwald
A, Boldrick JC, Sabet H, Tran T, Yu X, Powell JI,
Yang L, Marti GE, Moore T, Hudson J Jr, Lu L, Lew
is DB, Tibshirani R, Sherlock G, Chan WC, Greiner
TC, Weisenburger DD, Armitage JO, Warnke R, Staudt
LM, et al, Nature, 2000, 403, 503-11). Also the United States
Report on the reclassification and characterization of these cell lines by analyzing gene expression profiles for a panel of 60 cancer cell lines from the National Cancer Institute (Ross DT, Scherf U, Eisen MB, Perou CM, Rees C , S
pellman P, Iyer V, JeffreySS, Van de Rijn M, Walth
am M, Pergamenschikov A, Lee JC, Lashkari D, Shalo
n D, Myers TG, Weinstein JN, Botstein D, Brown PO,
Nat Genet, 2000, 24, 227-35), and a report discussing the relationship between the gene expression profiles of these 60 cancer cell line panels and the susceptibility of each cell line to various anticancer agents (Scherf U, Ross DT , Waltham M, Smith L
H, Lee JK, Tanabe L, Kohn KW, Reinhold WC, Myers T
G, Andrews DT, Scudiero DA, Eisen MB, Sausville E
A, PommierY, Botstein D, Brown PO, Weinstein JN, N
at Genet, 2000, 24, 236-44).

Similarly, using a DNA microarray (macro array using a part of a membrane filter),
There have been some reports on changes in gene expression that occur when tumor cells are treated with anti-cancer drugs (Rhee CH, R
uan S, Chen S, Chenchik A, Levin VA, Yung AW, Fulle
r GN, Zhang W, Oncol Rep, 1999, 6, 393-401. Zimm
ermann J, Erdmann D, Lalande I, Grossenbacher R, N
oorani M, Furst P, Oncogene, 2000, 19, 2913-20. Ku
doh K, Ramanna M, Ravatn R, Elkahloun AG, Bittner
ML, Meltzer PS, Trent JM, Dalton WS, Chin KV, Canc
er Res, 2000, 4161-6). These reports show that analysis of gene expression fluctuations reveals biological changes caused by comparison of characteristics of multiple cell populations, drug treatment, etc.
It is extremely useful for the purpose of comprehensive research at the molecular level.

[0009]

The object of the present invention is to treat tumor cells with E7070 and its related compounds,
It is to provide a surrogate marker for the antitumor effect of the compound.

[0010]

[Means for Solving the Problems] The present inventors analyzed by DNA microarray method the changes in gene expression caused by the action of E7070 and its related compounds on tumor cells sensitive to these anticancer agents. However, the inventors have found changes in gene expression that commonly appear with these anticancer agents.

Furthermore, it was found that some of these genes show changes in expression common to the three cancer types, and the changes in expression of these genes serve as surrogate markers for the antitumor effect of E7070 and its related compounds. The present invention has been completed by finding that it can be used. That is, the present invention provides the following.

1. 1). One or more tumor cells extracted from a cancer patient administered with an anticancer agent represented by the following general formula (I), selected from the group consisting of the genes shown in Tables 3 and 4 The expression level of the gene was measured, and 2). The expression level of the gene shown in Table 3 was increased as compared with tumor cells taken out from the cancer patient before administration of the anticancer drug,
Or assaying the susceptibility of the tumor cells to the anti-cancer drug, which comprises the step of determining that the tumor cells are susceptible to the anti-cancer drug when the expression level of the gene shown in Table 4 decreases. Method.

[0013]

[Chemical 3]

(In the formula, A ring is a monocyclic or bicyclic aromatic ring which may have a substituent, and B ring is a 6-membered ring which may have a substituent. Unsaturated hydrocarbon or an unsaturated 6-membered heterocycle containing one nitrogen atom as a heteroatom,
Ring C is a 5-membered heterocyclic ring which may have a substituent and contains 1 or 2 nitrogen atoms, W is a single bond or -CH =
The CH-, X is -N (R ¹⁾ - or an oxygen atom, Y is a carbon atom or a nitrogen atom, Z is -N (R ²⁾ - or a nitrogen atom, R ¹ and R ² are the same or different Means a hydrogen atom or a lower alkyl group. )

2. 1). A tumor cell taken out from a cancer patient is treated with an anticancer agent represented by the following general formula (I), and 2). A group of the tumor cell containing the genes shown in Tables 3 and 4 The expression level of one or more genes selected from 3). The expression level of the genes listed in Table 3 is increased, or the expression levels of the genes listed in Table 3 are increased as compared to untreated tumor cells. The method for assaying the sensitivity of the tumor cells to the anticancer drug, which comprises the step of determining that the tumor cells are susceptible to the anticancer drug when the expression level of the gene is decreased.

[0016]

[Chemical 4]

(In the formula, A ring is a monocyclic or bicyclic aromatic ring which may have a substituent, and B ring is a 6-membered cyclic group which may have a substituent. Unsaturated hydrocarbon or an unsaturated 6-membered heterocycle containing one nitrogen atom as a heteroatom,
Ring C is a 5-membered heterocyclic ring which may have a substituent and contains 1 or 2 nitrogen atoms, W is a single bond or -CH =
The CH-, X is -N (R ¹⁾ - or an oxygen atom, Y is a carbon atom or a nitrogen atom, Z is -N (R ²⁾ - or a nitrogen atom, R ¹ and R ² are the same or different Means a hydrogen atom or a lower alkyl group. )

3. The method according to 1 or 2, wherein the expression level of the gene is measured by quantifying RNA, which is a transcription product of the gene, by a DNA microarray.

4. 3. The method according to 1 or 2, wherein the expression level of the gene is measured by quantifying RNA, which is a transcription product of the gene, by quantitative PCR.

5. A RNA quantification reagent for use in the method according to 4, which comprises an oligonucleotide complementary to the RNA as a component.

6. The method according to 1 or 2, wherein the expression level of the gene is measured by quantifying a protein, which is a gene product of the gene, by an immunochemical method.

7. 7. The method according to 6, wherein the expression level of the gene is measured by quantifying the protein, which is the gene product of the gene, by ELISA.

8. 7. The method according to 6, wherein the expression level of the gene is measured by quantifying the protein, which is the gene product of the gene, by Western blotting.

9. An immunoassay reagent for use in the method according to 6, which comprises an antibody against the protein as a constituent element.

10. A ring is benzene or pyridine which may have a substituent, B ring is benzene which may have a substituent, and C ring is a pyrrole which may have a substituent. , W is a single bond, and X is
The method according to 1 or 2, wherein both and Z are -NH-.

[0026]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described in detail below.

In the assay method of the present invention, tumor cells are in vivo
Alternatively, it is characterized in that a change in the expression level of a specific gene in a tumor cell when exposed to an anticancer agent in vitro is used as an index of the sensitivity of the tumor cell to the anticancer agent.

Therefore, the assay method according to the first aspect of the present invention is 1). Tables 3 and 4 of tumor cells taken out from a cancer patient administered with an anticancer agent represented by the following general formula (I): The expression level of one or more genes selected from the group consisting of the genes listed in the table was measured, and 2). Compared with tumor cells taken out from the cancer patient before administration of the anticancer drug, the results are shown in Table 3. The method comprises the step of determining that the tumor cell is sensitive to the anti-cancer agent when the expression level of the described gene increases or the expression level of the gene described in Table 4 decreases.

The assay method according to the second aspect of the present invention is
1). A tumor cell taken out from a cancer patient is treated with an anticancer agent represented by the following general formula (I), and 2). A group of the tumor cell containing the genes shown in Tables 3 and 4 The expression level of one or more genes selected from 3). The expression level of the genes listed in Table 3 is increased, or the expression levels of the genes listed in Table 3 are increased as compared to untreated tumor cells. Determining that the tumor cell is susceptible to the anti-cancer agent when the expression level of the gene is decreased.

The anticancer agent in the present invention is a sulfonamide derivative or a sulfonate derivative represented by the above general formula (I).

In the above general formula (I), the meaning of ring A is
“A monocyclic or bicyclic aromatic group which may have a substituent
"Aromatic ring" means an aromatic hydrocarbon, or a nitrogen atom, an oxygen source.
Aromatics containing at least one of a child and a sulfur atom
It is a terror ring, and even if there are 1 to 3 substituents on the ring.
Show good things. Illustrates the main aromatic rings contained in ring A
And pyrrole, pyrazole, imidazole, thiophe
Amine, furan, thiazole, oxazole, benzene,
Lysine, pyrimidine, pyrazine, pyridazine, naphthale
Quinoline, isoquinoline, phthalazine, naphthyridine
, Quinoxaline, quinazoline, cinnoline, indo
Le, isoindole, indolizine, indazole,
Nzofuran, benzothiophene, benzoxazole,
Benzimidazole, benzopyrazole, benzothiazo
Etc. The aromatic ring has 1 to 3 substituents
If there are a plurality of substituents, the same or
It may be different. As the substituent, for example, a lower group
Substituted with an alkyl group or a lower cycloalkyl group
Good amino group, lower alkyl group, lower alkoxy group,
Hydroxyl group, nitro group, mercapto group, cyano group, lower alcohol
Kirthio group, halogen group, formula -ab [where a is a single bond
-(CH₂) K-, -O- (CH₂)_k-, -S-
(CH₂)_k-Or-N (R³)-(CH₂)_k-Is k
Is an integer of 1 to 5, R³Is a hydrogen atom or lower alkyl
Group, b is -CH₂-D (wherein d is a lower alkyl group)
Optionally substituted amino group, halogen group, hydroxyl group,
Lower alkylthio group, cyano group or lower alkoxy group
A group represented by the formula -a-e-
f [wherein a has the same meaning as described above, and e is -S (O)-or
Or-S (O)₂-, F is a lower alkyl group or lower
An amino group which may be substituted with an alkoxy group, a lower group
Alkyl group, trifluoromethyl group,-(CH₂)_m-B
Or -N (R^Four)-(CH₂)_m-B (where b is the same as above)
Has the same meaning, R^FourIs a hydrogen atom or a lower alkyl group
, M means an integer of 1 to 5)].
A group represented by formula -a-g-h [wherein a has the same meaning as described above]
, G is -C (O)-or -C (S)-, and h is low.
Amino group optionally substituted with a primary alkyl group, hydroxy
Group, lower alkyl group, lower alkoxy group,-(CH₂)_n
-B or -N (R^Five)-(CH₂)_n-B (where b is
Has the same meaning as above, R^FiveIs a hydrogen atom or lower alkyl
Kill group, n means an integer of 1 to 5]]
A group represented by formula -a-N (R⁶) -G-i [in the formula, a
And g have the same meanings as above, R⁶Is a hydrogen atom
Is a lower alkyl group, i is a hydrogen atom, a lower alkoxy group
Or f (where f has the same meaning as above)]
The group shown, formula -a-N (R ⁷) -E-f (in the formula, a,
e and f have the same meanings as described above, and R⁷Is a hydrogen atom
Or a lower alkyl group), or
Formula- (CH₂)_p-J- (CH₂)_q-B (where j is oxygen
Means an atom or a sulfur atom, and b has the same meaning as above.
However, p and q are the same or different and are integers of 1 to 5.
A group represented by (taste).

In the above substituent examples, when the amino group is substituted with two alkyl groups, these alkyl groups may be bonded to each other to form a 5- or 6-membered ring.
When ring A is a nitrogen-containing heterocycle having a hydroxyl group or a mercapto group, these groups may have a resonance structure to form an oxo group or a thioxo group.

The "6-membered unsaturated hydrocarbon optionally having a substituent or an unsaturated 6-membered heterocycle containing one nitrogen atom as a heteroatom", which means the ring B, is partially Benzene or pyridine, which may be hydrogenated, may have 1 or 2 substituents on the ring, and if there are 2 substituents, may be the same or different. Show.

The "5-membered heterocycle containing 1 or 2 nitrogen atoms, which may have a substituent (s)", which means ring C, means
Pyrrole, pyrazole, imidazole, which may be partially hydrogenated, and the substituent 1 or 2 on the ring.
And the number of substituents is the same or different.

Examples of the substituent which ring B and ring C may have include, for example, halogen group, cyano group, lower alkyl group, lower alkoxy group, hydroxyl group, oxo group, and formula -C.
(O) -r (in the formula, r represents a hydrogen atom, an amino group which may be substituted with a lower alkyl group, a lower alkyl group, a lower alkoxy group or a hydroxyl group), or a lower alkyl group Good amino group, trifluoromethyl group and the like can be mentioned.

In the above general formula (I), R ¹ and R ² and the lower alkyl group in the definition of the substituent which ring A, ring B and ring C may have are a straight-chain alkyl group having 1 to 6 carbon atoms. Chain or branched alkyl groups such as methyl, ethyl, n
-Propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, n-pentyl group (amyl group), isopentyl group, neopentyl group,
tert-pentyl group, 1-methylbutyl group, 2-methylbutyl group, 1,2-dimethylpropyl group, n-hexyl group, isohexyl group, 1-methylpentyl group, 2-methylpentyl group, 3-methylpentyl group, 1 , 1-dimethylbutyl group, 1,2-dimethylbutyl group, 2,2-dimethylbutyl group, 1,3-dimethylbutyl group, 2,3-dimethylbutyl group, 3,3-dimethylbutyl group, 1-ethylbutyl Group, 2-ethylbutyl group, 1,1,2-trimethylpropyl group, 1,2,2-trimethylpropyl group,
1-ethyl-1-methylpropyl group, 1-ethyl-2-
It means a methylpropyl group or the like. Of these, preferred groups include a methyl group, an ethyl group, an n-propyl group,
An isopropyl group, an n-butyl group, an isobutyl group and the like can be mentioned, and among these, a methyl group, an ethyl group, an n-propyl group and an isopropyl group can be mentioned as the most preferable group.

The lower cycloalkyl group in the definition of the substituent which the ring A may have means a cycloalkyl group having 3 to 8 carbon atoms, and examples thereof include a cyclopropyl group, a cyclopentyl group and a cyclohexyl group. be able to.

The lower alkoxy group in the definition of the substituent which ring A, ring B and ring C may have is a methoxy group,
Ethoxy group, n-propoxy group, isopropoxy group, n
It means an alkoxy group derived from the above lower alkyl group such as -butoxy group, isobutoxy group and tert-butoxy group, and the most preferable group among these is methoxy group and ethoxy group. The lower alkylthio group means an alkylthio group derived from the above lower alkyl group. Further, examples of the halogen atom include a fluorine atom, a chlorine atom and a bromine atom.

The sulfonamide derivative or sulfonate derivative represented by the general formula (I) may form a salt with an acid or a base. The anticancer agent in the present invention also includes a salt of the compound represented by the general formula (I). Examples of the salt with an acid include inorganic acid salts such as hydrochloride, hydrobromide and sulfate, acetic acid, lactic acid, succinic acid, fumaric acid, maleic acid, citric acid, benzoic acid, methanesulfonic acid, p-
Mention may be made of salts with organic acids such as toluenesulfonic acid. Examples of the salt with a base include inorganic salts such as sodium salt, potassium salt and calcium salt, and salts with organic base such as triethylamine, arginine and lysine.

It goes without saying that not only hydrates of these compounds but also all optical isomers are included when they exist. Further, the anticancer agent of the present invention includes a compound that exhibits a strong antitumor activity, but exhibits an antitumor activity by being metabolized in vivo by metabolism such as oxidation, reduction, hydrolysis and conjugation. Furthermore, the anticancer agent in the present invention also includes a compound which undergoes metabolism such as oxidation, reduction and hydrolysis in vivo to produce the compound represented by the general formula (I).

The tumor cells to be tested for sensitivity are not particularly limited as long as they show sensitivity to the anticancer drug represented by the general formula (I). Examples thereof include tumor cells derived from colon cancer, lung cancer, breast cancer, leukemia, pancreatic cancer, kidney cancer, melanoma, malignant lymphoma, head and neck cancer, gastric cancer and the like.

The tumor cells taken out from the cancer patient also include the tumor cells contained in the cancer tissue extracted from the cancer patient.

In the case of the assay method of the first aspect, the tumor cells taken out from a cancer patient are administered with an anticancer agent represented by the general formula (I) in an amount such that the sensitivity of the tumor cells can be measured. Tumor cells taken from a living cancer patient,
Tumor cells removed from a cancer patient, usually administered at a dose of 100-1500 mg for 1-14 days.

In the case of the assay method of the second aspect, the sensitivity of tumor cells can be measured under the condition that the anticancer agent represented by the general formula (I) is allowed to act on the tumor cells taken out from the cancer patient. Anything can be used, usually 0.01 to 10 in the medium.
A condition of culturing for 6 to 72 hours at an anticancer agent concentration of μM can be mentioned.

The expression level of a gene can be measured by quantifying RNA, which is a transcription product of a gene, or protein, which is a gene product. Quantification of RNA or protein can usually be performed by extracting RNA or protein from tumor cells and quantifying RNA or protein in the extract. Below, 1. RNA or protein extraction, 2. RNA quantification, 3. These examples will be described in detail in the order of protein quantification.

1. Extraction of RNA 1). Extraction of RNA or Protein from Cancer Tissue of Patients Receiving Anticancer Agent Represented by General Formula (I) From patients administered anticancer agent represented by General Formula (I),
RNA or protein is extracted from the cancer tissue extracted by biopsy or the like by the method described below.

RNA may be extracted according to a general RNA extraction method. For example, TRIZOL reagent (Lifetech Oriental) or the like may be used according to the attached operation method.
Specifically, it is as follows. 50 ml of cancer tissue is added with 1 ml of TRIZOL reagent and homogenized using a Teflon (registered trademark) homogenizer. Centrifuge this (1
(2,000 xg, 10 minutes, 4 ° C), and leave the obtained supernatant at room temperature for 5 minutes, and then add chloroform at a ratio of 0.2 ml to 1 ml of the TRIZOL reagent used. This solution is shaken vigorously for 15 seconds, stirred, left at room temperature for 2-3 minutes, and then centrifuged (12,0
00 xg, 15 minutes, 4 ° C). After centrifugation, transfer the aqueous layer to a new tube, add 0.5 ml of isopropyl alcohol to 1 ml of TRIZOL reagent used, leave it at room temperature for 10 minutes, and then centrifuge (12,000 xg, 10 minutes, 4 ° C). . The obtained precipitate was washed with 75% ethanol and then air-dried to obtain total RNA.
To be used for the subsequent operations.

Extraction of proteins from cancer tissue is described by Bollag, D.
M., Rozycki MD, Edelstein SJ, Protein Method
s, 1996, Wiley-Liss, Inc., New York, USA Walke
r, JM, The protein handbook, 1996, Humana Press,
It may be performed according to the method described in New Jersey, USA and the like.

2). Extraction of RNA or protein from cancer cells cultivated in the presence of the anticancer agent represented by the general formula (I) From a cancer tissue obtained by biopsy from a patient, cancer cells (tumor Cells). For example, Hamburger et al. (Hamburger A., Salmon SE, Kim MB, Trent J.
M., Soehnlen BJ, Alberts DS, and Schmidt H.
T., Cancer Res., 38, 3438-3443, 1978), aseptically cut the obtained tissue, and then use a stainless mesh, an injection needle, a nylon mesh, etc. to suspend the cell suspension. Prepare. The cells thus obtained were mixed with an appropriate medium (for example, RPMI-1640 containing 10 to 15% FCS, MEM, McCoy
Culture the medium). The obtained cancer cells are treated in the presence of the anticancer agent represented by the general formula (I) for a suitable period of time, preferably 3 hours, 6 hours, 12 hours or 24 hours, more preferably 12 hours.
After culturing at 37 ° C. under 5% CO _{2 for an} hour, RNA or protein is extracted by the method described below. In addition, the soft agar culture method (Hamburge
r A., and Salmon SE, Science, 197, 461-463,197
7, Hamburger A., and Salmon SE, J. Clin. Inves
t., 60, 846-854, 1977, Von Hoff DD, and Johnso
n, GE, Proc. Am. Assoc. Cancer Res., 20, 51, 19
It is also possible to specifically separate and use only cancer cells by using 79).

Extraction of RNA from cancer cells can be performed by extracting R from cancer tissue.
Similar to the extraction of NA, it may be performed according to a general RNA extraction method. For example, when TRIZOL reagent (Lifetech Oriental) is used, it may be performed according to the attached operation method. Specifically, 1 ml of TRIZOL reagent is added to ⁵ to 10 × 10 ⁶ cancer cells, and then the same operation as RNA extraction from cancer tissue may be performed.

The extraction of proteins from cancer cells may be performed according to the method described in the textbook, as in the case of extraction from cancer tissues.

2. Quantification of RNA RNA is analyzed by Northern blot analysis, DNA microarray, RT-P
It can be quantified by techniques such as CR and quantitative PCR. Preferably D
NA microarray / quantitative PCR is preferable.
Each will be described below, but the present invention is not limited thereto.

Quantitation by DNA microarray is performed as follows. SuperScript C using the first RNA obtained as a template
hoice System (Life Tech Oriental) and T7-d
(T) ₂₄ primer is used to synthesize double-stranded cDNA, and then biotinylated cRNA is synthesized using the cDNA as a template.

Specifically, first, from the RNA obtained, T7-d (T)
Synthesize single-stranded DNA using ₂₄ primers, then dNT
Double-stranded cD by adding P ・ DNA ligase ・ DNA polymerase I ・ RNase H and then adding T4 DNA polymerase I
Synthesize NA. After purifying the obtained cDNA, RNA Transcript
Using the Labeling Kit (Enzo Diagnostics), the labeling reaction is performed by adding biotinylated UTP and CTP. After purification of the reaction product, it is heated in 200 mM Tris-acetate pH8.1, 150 mM magnesium acetate, 50 mM potassium acetate at 94 ° C for 35 minutes to obtain fragmented cRNA.

Fragmented cRNA was isolated, for example, from 100 mM MES, 1
M sodium salt, 20 mM EDTA, 0.01% Tween 20, 45 ° C
Hybridize for 16 hours with GeneChip (Affymetrix) Hu6800 or equivalent product. After hybridizing,
GeneChip is washed and stained according to the protocol EukGE-WS2 attached to the Affymetrix fluidics station. For staining, streptavidin-phycoerythrin and biotinylated anti-streptavidin goat antibody are used. GeneChip after staining
HP Argon Ion Laser Confocal Microscope (Hewlett P
ackard) to measure fluorescence intensity. In the case of this fluorescent dye, the measurement was performed using excitation light of 488 nm and 570 nm.
To measure the fluorescence of.

Quantitative data analysis, preferably GeneChip
This is done using software (Affymetrix). In order to perform RNA quantification, the difference ([perfect match hybridization signal (perfec
t match hybridization signal)]-[mismatch signal]) "
e) is obtained, and when this value is 50 or more and the quantitative value of RNA deviates between the two conditions, preferably when dissociating 1.8 times or more, the expression of the gene is significantly " It is judged to have increased or decreased.

One or more of the genes listed in Table 3
When the amount of RNA increases or the amount of RNA of one or more genes listed in Table 4 decreases, the tumor cell is determined to be sensitive to the anticancer drug.

Quantitative PCR was performed using SYBR Green and ABI Prism.
7700 Sequence Detection System (Perkin-Elmer Appl
ied Biosystems) as follows.

The operation is performed in two steps of reverse transcription reaction and PCR reaction. The first step, reverse transcription, involves dNT
P ・ oligo d (T) ₁₆ primer ・ Rnase inhibitor ・ Mult
iscribe Reverse Transcriptase (Perkin-Elmer Applie
d Biosystems) is added, the mixture is kept at 25 ° C for 10 minutes, and then heated at 48 ° C for 30 minutes. The reaction is stopped by heating at 95 ° C for 5 minutes.

The obtained cDNA is subjected to the PCR reaction of the second stage. For PCR reaction, for example, 4 ng cDNA, 1xSYBR PCR buffer, 3 mM MgCl ₂ , 200 μM each dATP, dCTP, dGTP, 400 μM
M dUTP, 200 nM primer pair, 0.01 U / μl AmpErase U
NG, 0.025 U / μl AmpliTaq Gold DNA Polymerase (Perk
in-Elmer Applied Biosystems). The reaction conditions are, for example, 50 ° C for 2 minutes, 95 ° C for 10 minutes, and then 95 ° C.
Perform 40 cycles of 20 seconds, 55 ° C, 20 seconds, 72 ° C, 30 seconds. Primers and probes are, for example, Primer Expressio
Design using n (Perkin-Elmer Applied Biosystems). The comparison of a plurality of samples is performed by correcting the quantitative value with a house keeping gene, preferably GAPDH mRNA level, in which the transcription amount of each sample has little variation.

Of one or more of the genes listed in Table 3
When the amount of RNA increases or the amount of RNA of one or more genes listed in Table 4 decreases, the tumor cell is determined to be sensitive to the anticancer drug.

The present invention also provides a RNA quantification reagent for use in the assay method of the present invention, comprising as a constituent an oligonucleotide complementary to RNA which is a transcription product of a symmetrical gene whose expression level is to be measured. To do. The constituent oligonucleotides are primers and / or probes used in quantitative PCR and can be designed as described above. The RNA quantification reagent of the present invention may contain, in addition to the above-mentioned oligonucleotide, components commonly used in general quantification reagents.

3. Quantification of protein Protein is quantified based on its activity or antigenicity.
It is preferable to use a quantitative assay based on an antigenic property that is generally applicable to proteins, that is, an immunochemical assay.

The antibody against the protein was reported by Parker et al. From its amino acid sequence (Parker JMR, Guo D., Hodges.
RS, Biochemistry, 25,5425, 1986) or Karpl
Report from us et al. (Karplus PA, Schulz GE, Naturwiss
Enschaften, 72, 212, 1985) to predict an antigenic determinant, synthesize a peptide, or express a fusion protein such as a fusion protein with glutathione synthase (GST) and purify it with a glutathione column to purify the antigen. Rabbits, mice, etc. are immunized with the obtained antigens, and polyclonal, preferably monoclonal antibodies (Harlow E., Lane D., An
tibodies: ALaboratory Manual, 1988, Cold Spring Ha
rbor Laboratory Press, New York) can be produced. In addition, if a commercially available antibody is available, it can be used after confirming its specificity.

Using the obtained antibody, for example, an enzyme immunoassay (Eiji Ishikawa et al., Ikusho Shoin, 1982) or Enzyme Immun
oassay (Ishikawa E., Kawai T., Miyai, K., Igaku-Sh
ELISA, by the method described in Oin, Tokyo New York, 1981).
Alternatively, RIA is performed to quantify the protein. When the protein is secreted outside the tumor cells, it is possible to quantify the protein in the medium without extracting the protein from the tumor cells.

When the protein amount of one or more genes shown in Table 3 is increased or the protein amount of one or more genes shown in Table 4 is decreased, the tumor cells are treated with the anticancer agent. To be sensitive to.

The present invention also provides an immunoassay reagent containing an antibody against the protein as a constituent for use in the assay method of the present invention. The antibody as a component can be obtained as described above. The immunoassay reagent of the present invention may contain components commonly used in general immunoassay reagents, in addition to the above-mentioned antibody.

[0068]

EXAMPLES The present invention will be described below with reference to specific examples, but the present invention is not limited thereto.

[0069]

Example 1 Culture of E7070-sensitive strain and resistant strain and extraction of RNA All cells were RPMI-supplemented with 10% fetal bovine serum, 100 units / ml penicillin, and 100 mg / ml streptomycin.
The cells were cultured in 1640 medium at 37 ° C. under 5% CO ₂ .

E7070 or ER-35748 whose structural formula is shown below
Alternatively, ER-68487 is used as an E7070 sensitive strain HCT116-C9 and E7070
The cells were added at a concentration of 8 μM to the medium of the resistant strain HCT116-C9-C1 and cultured, and the cells after 0, 3, 6, 12 hours were collected. Similarly, cells cultured for 12 hours without adding a drug were also collected.

[0071]

[Chemical 5]

Total RNA was extracted from these cells and subjected to the subsequent analysis. RN extracted from cells 12 hours after drug addition
RNA extracted from cells cultured for 12 hours without addition of A and a drug was used for gene expression analysis by the DNA microarray described in Example 2. HCT116-C9 is human colon cancer-derived HCT116 (American Type Culture Collection, Mana
ssas, VA, USA)
HCT116-C9-C1 is an E7070-resistant substrain obtained by culturing 16-C9 in the presence of E7070 and gradually increasing the E7070 concentration.

Similarly, E7070 was replaced with E7070 sensitive strains LX-1 and E707.
Add to the culture medium of 0-resistant strain LX-1-E2 at a concentration of 8 μM and culture,
The cells after 0, 3, 6 and 12 hours were collected. Total RNA was extracted from these cells and subjected to subsequent analysis. LX-1 (C
ancer Chemotherapy Center, Japan Foundation for Can
cer Research, Tokyo, Japan) is a cell line derived from human small cell lung cancer. E7070 resistant substrain obtained by culturing this LX-1 in the presence of E7070 and gradually increasing the E7070 concentration was LX-1. -It's E2.

Cell lines used HCT116-C9, HCT116-C9-C
1, LX-1, LX-1-E2 with E7070 added and cultured for 72 hours, MTT method (Mosmann T., J. Immunol. Methods, 65, 55, 1983)
The cell growth inhibition curve measured by the method is shown in FIG. Actual operation is CellTiter 96 Non-Radioactive Cell Proliferat
Ion Assay (Promega, Madison, WI) was used according to the attached procedure.

Extraction of total RNA from the recovered cells was performed using TRIZO
The procedure was carried out using L reagent (Lifetech Oriental) according to the attached procedure.

[0076]

[Example 2] Gene expression analysis by DNA microarray 1). CDNA synthesis and biotin labeling The RNA obtained in Example 1 was dissolved in 100 μl of sterile water treated with diethylpyrocarbonate (DEPC), and further RNea
Purified using sy column (QIAGEN), SuperScript Choi
ce System (Life Tech Oriental) and T7-d (T) ₂₄
Double-stranded cDNA was synthesized using the primers.

First, 10 μg of RNA was added to 5 μM of T7-d (T) ₂₄ primer, 1x First strand buffer, 10 mM DTT, and 500 μM of dN.
TP mix, 20 units / μl SuperScript II Reverse Trans
Criptase was added and reacted at 42 ° C for 1 hour to synthesize single-stranded DNA. Then 1x Second strand buffer, 200 μM dNT
P mix, 67 U / ml DNA ligase, 270 U / ml DNA polymerase I and 13 U / ml RNase H were added and reacted at 16 ° C for 2 hours to synthesize double-stranded cDNA. Further, 67 U / ml T4 DNA polymerase I was added and reacted at 16 ° C for 5 minutes.
The reaction was stopped by adding l of 0.5 M EDTA.

The obtained cDNA was purified with phenol / chloroform, and RNA Transcript Labeling Kit (Enzo Di
agnostics) and a labeling reaction with biotinylated UTP and CTP was performed according to the attached procedure. After purifying the reaction product with Rneasy column, 200 mM Tris-acetate pH
The cRNA was fragmented by heating in 8.1, 150 mM magnesium acetate, 50 mM potassium acetate at 94 ° C. for 35 minutes.

2). Hybridization to DNA microarray (GeneChip) and measurement Fragmented cRNA was treated with 100 mM MES, 1 M sodium salt, 20 mM.
16 hours at 45 ° C in mM EDTA, 0.01% Tween 20, GeneCh
Hybridized to ip (Affymetrix) Hu6800. After hybridization, GeneChip will be Affymetrix fluidics statio
The cells were washed and stained according to the protocol EukGE-WS2 attached to n. For staining, streptavidin-phycoerythrin and biotinylated anti-streptavidin goat antibody were used. The stained GeneChip was scanned using an HP argon ion laser confocal microscope (Hewlett Packard), and the fluorescence intensity was measured. The measurement was performed with excitation light of 488 nm and fluorescence of 570 nm.

GeneChip softwar is used for all quantitative data analysis.
e (Affymetrix). To perform RNA quantification, the difference ([perfect match hybridization signal (perfect match h
ybridization signal)]-[Mismatch signal (mismat
ch signal)]) ”, the average (average difference) is calculated, and when this value is 50 or more and the quantitative value of RNA deviates between two conditions, preferably 1.8 times or more dissociation Therefore, it was judged that the expression of the gene was significantly “increased” or “decreased”.

E7070 or ER-35748, ER-68487 to HCT116
-C9 medium was added at a concentration of 8 μM and cultured, and 12 hours later, the results of analysis by GeneChip of RNA extracted from the cells were obtained by the same procedure without adding a drug, and the RCT of HCT116-C9 cells was obtained.
It was compared with the NA analysis results. As a result, E7070, ER-35748,
Table 1 shows a list of genes whose expression was commonly increased by the treatment of 3 agents of ER-68487 (also showing the GenBank accession numbers (same in the table below)). Table 2 shows a list of genes whose expression was commonly reduced by the treatment with the drug.

[0082]

[Table 1]

[0083]

[Table 2]

[0084]

[Table 3]

[0085]

[Table 4]

[0086]

[Table 5]

[0087]

[Table 6]

Regarding the genes shown in Tables 1 and 2,
Using two E7070 resistant strains (C9-C1 and another resistant strain (C9-C4) obtained by the same method as C9-C1),
After 12 h treatment with μM E7070
It was analyzed by eChip. As a result, a gene whose expression was increased or decreased 1.8 times or more was not observed in both strains.

From the above results, it is possible to use, as a surrogate marker for the antitumor effect of E7070 and its related compounds, by measuring the changes in the genes that commonly change due to the treatment with these three anticancer agents, alone or in combination. it is obvious.

[0090]

Example 3 Analysis of Gene Expression by Quantitative PCR The expression variation of the gene obtained in Example 2 was due to the fact that E7 of tumor cells was
For the purpose of confirming that it reflects the sensitivity to 070, the RNA shown in Example 1 was used to detect changes in the expression of these genes in E7070-sensitive cells and resistant cells.
Green and ABI Prism 7700 Sequence Detection System
(Perkin-Elmer Applied Biosystems)
It was examined by CR.

The operation was performed in two steps of reverse transcription reaction and PCR reaction. The first step, the reverse transcription reaction, required 1 μg of total RNA for 1
x TaqMan RT buffer, 5.5 mM MgCl ₂ , 500 μM dNTP mi
x, 2.5 μM oligo d (T) ₁₆ primer, 0.4 U / μl RNase
Inhibitor, 1.25 U / μl Multiscribe Reverse Transcr
Add iptase (Perkin-Elmer Applied Biosystems) and add 2
After keeping the temperature at 5 ° C for 10 minutes, heating was performed at 48 ° C for 30 minutes. The reaction was stopped by heating at 95 ° C for 5 minutes.

The obtained cDNA was subjected to the second stage PCR reaction. PCR reaction is 4 ng cDNA, 1xSYBR PCR buffer, 3 mM
MgCl ₂ , 200 μM dATP, dCTP, dGTP, 400 μM dUTP, 20 each
0 nM primer pair, 0.01 U / μl AmpErase UNG, 0.025 U
/ μl AmpliTaq Gold DNA Polymerase (Perkin-Elmer Ap
plied Biosystems). Reaction conditions are 50 ℃
Two cycles of 95 ° C for 10 minutes, 95 ° C for 20 seconds, 55 ° C for 20 seconds, and 72 ° C for 30 seconds were performed for 40 cycles. The primer is G
APDH is SEQ ID NOS: 1 and 2, S80343 is SEQ ID NOS: 3 and 4,
U07919 is SEQ ID NO: 5 and 6, U11791 is SEQ ID NO: 7 and 8, M95809 is SEQ ID NO: 9 and 10, U18291 is SEQ ID NO: 1.
1 and 12, U63743 used the sequence number 13 and 14, and M61764 used the oligonucleotide which has the base sequence shown in sequence number 15 and 16.

The amount of mRNA in each sample was quantified by measuring the fluorescence intensity. In addition, in the comparison of multiple samples,
The quantitative value was corrected with the amount of GAPDH mRNA of each sample. Control the quantitative value at 0 hours of drug treatment (100
%), The relationship between the drug treatment time and the amount of each mRNA is shown in FIGS. Each gene has the most sensitive HC
T116-C9 showed the largest expression fluctuation (Fig. 2), and the sensitive strain LX-
1 has a smaller fluctuation range than HCT116-C9, but S80343
Expression variation was shown in all genes except (Fig. 3). On the other hand, in the resistant strain HCT116-C9-C1, no change in expression was observed (Fig. 4), and in the resistant strain LX-1-E2, which had some effect of suppressing the growth at the drug concentration of 8 µM used in the experiment. , U11791
Shows the expression variation, and a slight expression variation was observed in M95809 and U18291 (Fig. 5).

The expression variation of the gene shown in Example 2 was E7.
It was confirmed that the gene shown in Example 2, which is correlated with the sensitivity to 070, can be used as a surrogate marker for the antitumor effect of E7070 and its related compounds by measuring the change alone or in combination.

[0095]

[Example 4] Analysis by ELISA Among the genes shown in Table 2 of Example 2, X51956 (Human ENO2 gene), which is reported to be secreted extracellularly
ELISA method already reported for for neuron specific (gamma) enolase (Duncan ME, McAleese S.
M., Booth NA, Melvin WT, and Fothergill J.
E., J. of Immunol. Methods, 151, 227-236, 1992, Ya
maguchi K., Aoyagi K., Urakami K., Fukutani T., Ma
ki N., Yamamoto S., Otsubo K., Miyake Y., and Koda
ma T., Jpn. J. Cancer Res., 86, 698-705, 1995) to examine the expression level. The NSE ELISA kit from Eiken Kagaku (Tokyo) was used for the actual measurement, and the operation was according to the attached data.

[0096]

Example 5 Gene Expression Analysis in Three Cancer Types HCT116-C9 (human colon cancer cell line), MDA-MB-435 (human breast cancer cell line) and MOLT-4 (human T lymphoblastic leukemia cell line) For the three types of cancer, E7070 in the same manner as in Example 2.
Gene Chip changes caused by (8 μM treatment for 12 h)
It investigated using.

Table 3 shows a list of genes whose expression is increased by 1.8 cultures or more in the three cancer types. In addition, Table 4 shows a list of genes whose expression is suppressed in all three cancer types by 1.8 times or more. Genes whose expression is commonly changed (enhanced or decreased) in these three cancer types established from cancer patients with completely different genetic backgrounds may be deeply related to the antitumor action mechanism of E7070 common to tumor cells. Therefore, it is considered that it can be an effective marker when assaying the sensitivity of tumor cells to E7070 and its related compounds.

[0098]

[Table 7]

[0099]

[Table 8]

[0100]

[Table 9]

[0101]

[Table 10]

[0102]

INDUSTRIAL APPLICABILITY According to the present invention, tumor cells taken out from a cancer patient to which an anticancer agent represented by the general formula (I) is administered,
By measuring the expression levels of the genes shown in Tables 3 and 4, or by allowing the anti-cancer agent represented by the general formula (I) to act on the tumor cells taken out from the cancer patient, By measuring the expression levels of the genes listed in the table,
It becomes possible to examine the sensitivity of the tumor cells to the anticancer drug.

[0103]

[Sequence list] <110> Eisai Co., Ltd. <120> Method for assaying sensitivity of tumor cells to anticancer drug <130> P-9401 <150> JP 2000-357398 <151> 2000-11-24 <160> 16 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 gaaggtgaag gtcggagtc 19 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 gaagatggtg atgggatttc 20 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 gcattttacg gttccctgag at 22 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 gatacgccac atgttcacct tc 22 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 cagaatcaat agcccagaga gctt 24 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 gttgtggcgt tagaagattg gatc 24 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 gtcattctgc tgagcttgca ctta 24 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 gagagattct accaggtcgt catca 25 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 ccaagttacg aagctctgtc cat 23 <210> 10 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 tgtaggctgt ctggagcatc tct 23 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 tgttgattcc tcagaacgca tc 22 <210> 12 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 tgtatcatct cgcctaagac caag 24 <210> 13 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 atctcaccag gcataagctc ct 22 <210> 14 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 acagttcctc ctcttccttg gat 23 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 ctcaagaggc tgacgcagaa t 21 <210> 16 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 ctggctgaca tgatggtaga cac 23

[Brief description of drawings]

FIG. 1 Cell lines HCT116-C9, HCT116-C9-C1, LX-1, LX-
The cell growth suppression curve by E7070 of 1-E2 is shown.

FIG. 2 shows the results of quantitative PCR analysis of gene expression fluctuations in the E7070-sensitive strain HCT116-C9.

FIG. 3 shows the results of quantitative PCR analysis of gene expression fluctuations in the E7070-sensitive strain LX-1.

FIG. 4 shows the results of quantitative PCR analysis of gene expression fluctuations in the E7070 resistant strain HCT116-C9-C1.

FIG. 5 shows the results of quantitative PCR analysis of gene expression changes in the E7070 resistant strain LX-1-E2.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 37/00 102 C12N 15/00 A (72) Inventor Takatoshi Kawai 2-11-33 East, Tsukuba, Ibaraki ( 72) Inventor Hiroyuki Kato 5-25-41 Goshogaoka, Moriya-machi, Kitasoma-gun, Ibaraki Prefecture (72) Takeshi Nagasu 85-23, Nagakuni, Tsuchiura-shi, Ibaraki F-term (reference) 4B024 AA12 CA09 HA14 4B063 QA07 QA19 QQ08 QQ43 QQ52 QR32 QR35 QR55 QR62 QR77 QR84 QS25 QS34 QX02

Claims (10)

[Claims] 1. A tumor cell extracted from a cancer patient administered with an anticancer agent represented by the following general formula (I), selected from the group consisting of the genes shown in Tables 3 and 4. 1
2). The expression level of the gene shown in Table 3 is increased as compared with the tumor cells taken out from the cancer patient before administration of the anticancer drug,
Or assaying the susceptibility of the tumor cells to the anti-cancer drug, which comprises the step of determining that the tumor cells are susceptible to the anti-cancer drug when the expression level of the gene shown in Table 4 decreases. Method. [Chemical 1] (In the formula, ring A is a monocyclic or bicyclic aromatic ring which may have a substituent, and ring B is a 6-membered unsaturated carbon ring which may have a substituent. An unsaturated 6-membered heterocycle containing one nitrogen atom as hydrogen or a heteroatom, and C ring containing one or two nitrogen atoms which may have a substituent 5
Membered heterocycle, W is a single bond or -CH = CH-, X
It is -N (R ¹⁾ - or an oxygen atom, Y is a carbon atom or a nitrogen atom, Z is -N (R ²⁾ - or a nitrogen atom,
R ¹ and R ² are the same or different and each represents a hydrogen atom or a lower alkyl group. ) 2. A tumor cell taken out from a cancer patient is treated with an anticancer agent represented by the following general formula (I), and 2). The tumor cell is shown in Tables 3 and 4. The expression level of one or more genes selected from the group consisting of 3) genes is measured, and 3). The expression level of the genes listed in Table 3 is increased as compared to untreated tumor cells, or A method for assaying the susceptibility of the tumor cell to the anticancer drug, comprising the step of determining that the tumor cell is susceptible to the anticancer drug when the expression level of the gene shown in Table 4 decreases. . [Chemical 2] (In the formula, ring A is a monocyclic or bicyclic aromatic ring which may have a substituent, and ring B is a 6-membered unsaturated carbon ring which may have a substituent. An unsaturated 6-membered heterocycle containing one nitrogen atom as hydrogen or a heteroatom, and C ring containing one or two nitrogen atoms which may have a substituent 5
Membered heterocycle, W is a single bond or -CH = CH-, X
It is -N (R ¹⁾ - or an oxygen atom, Y is a carbon atom or a nitrogen atom, Z is -N (R ²⁾ - or a nitrogen atom,
R ¹ and R ² are the same or different and each represents a hydrogen atom or a lower alkyl group. ) 3. The method according to claim 1, wherein the expression level of the gene is measured by quantifying RNA, which is a transcription product of the gene, by a DNA microarray. 4. The method according to claim 1, wherein the expression level of the gene is measured by quantifying RNA, which is a transcription product of the gene, by quantitative PCR. 5. An RNA quantification reagent, which comprises an oligonucleotide complementary to said RNA as a constituent, for use in the method according to claim 4. 6. The method according to claim 1, wherein the expression level of the gene is measured by quantifying a protein, which is a gene product of the gene, by an immunochemical method. 7. The method according to claim 6, wherein the expression level of the gene is measured by quantifying the protein, which is the gene product of the gene, by ELISA. 8. The method according to claim 6, wherein the expression level of the gene is measured by quantifying the protein, which is the gene product of the gene, by Western blotting. 9. An immunoassay reagent for use in the method according to claim 6, which comprises an antibody against the protein as a constituent element. 10. A ring A is optionally substituted benzene or pyridine, a ring B is optionally substituted benzene, and a ring C is optionally substituted. Or pyrrole, W is a single bond, and both X and Z are -NH-.
The method described in.