JP2003284594A - Method for detecting ovarian cancer cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for detecting ovarian cancer cells, by utilizing abnormality in a gene level as an indicator. <P>SOLUTION: This method for detecting the ovarian cancer cells comprises (1) a process in which an expression level of at least one ovarian cancer-related gene selected from genes having specified base sequences registered in Genbank or their authlogs is measured in a test specimen and (2) another process in which the measured value of the expression level of the gene obtained in the process (1) is compared with a control value of the expression level of the gene so that the ovarian cancer cells in the test specimen are detected based on a difference between the values. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for detecting ovarian cancer cells.

[0002]

BACKGROUND OF THE INVENTION Although it is gradually becoming clear that cancer is a disease caused by a gene-related abnormality, the mortality rate of cancer patients is still high, and it is currently available. It indicates that the diagnostic and therapeutic methods are not fully satisfactory. Therefore, it has been earnestly desired to develop a method for detecting cancer cells using an abnormality at a gene level as an index for use in early detection of cancer, rapid diagnosis, evaluation of treatment methods, and the like.

[0003]

Means for Solving the Problems Under the circumstances, the present inventors have conducted extensive studies, and as a result, found that the expression level of a specific gene in ovarian cancer tissue was different from that in normal tissue, and the present invention I arrived. That is, the present invention is: A method for detecting ovarian cancer cells, which comprises (1) one or more genes selected from genes having orthologs having a nucleotide sequence registered in Genbank with any of the following registration numbers 1 or 2 Measuring the expression level of said gene in a sample, (2) comparing the measured value of the expression level of the gene obtained in said step (1) with a control value of the expression level of said gene, and based on the difference, said A method characterized by including a step of detecting ovarian cancer cells in a sample (hereinafter sometimes referred to as the detection method of the present invention), <Registration number group 1> X02344, AI884621, X59932, AI98943.
2, X74929, AI346658, AI354351, AI332641, U28964, A
I672414, D26598, H44597, U65785, AA775776, AA74243
8, AI887311, M13560, AI807277, D38047, AI798743, X
04366, AI791182, H15313, AA004221, L24521, AI95497
5, D45248, AI973186, AF051782, AI762891, N37049, A
A554681, X57346, W21894, U48705, AA112121, AL05029
0, AW024537, W60640, AW024572, M26252, AA020846, U
85611, R54450, U78095, AI983181, AI741591, AI99112
2, W28869, AI147040, HG1614-HT1614, AI986430, AJ00
2308, AA062899, AA532392, AI560151, Y15915, AI3010
60, HG1980-HT2023, AI351790, M21186, AL041566, AI8
31655, AA211690, AI885170, AI241578, M91670, AW020
965, D42123, N32858, AI460118, AI539386, AI62038
1, AI804075, U21689, R20554, D89052, AI684451, AA4
79496, AA131307, X57348, AI992284, U77604, AI66041
2, AF054174, AI878836, AI991845, AW001374, AF00539
2, AI862775, U47634, AA928646, M83751, AI341234, A
I018296, AI394307, Z69043, AI032386, X00734, AI970
195, U49278, AI741313, AI634570, AI733988, AJ13118
2, AI613214, X67325, AW007442, AI653621, AA27853
8, T15777, AA543084, J03909, AA587236, L16842, AI5
57649, M34423, AI309320, AA622029, AI934540, Z9393
0, AA383718, X69699, AI979335, M65028, AA044793, A
I357616, AI417084, AF047436, AW009649, AW004750, A
I990317, AW007731, AI087923, AI867409, AI660570, A
F001601, N63815, AA004274, AI927408, K03515, AI624
028, AA576813, AI627731, M61832, AI923108, AW00950
5, AI969418, X73478, AI589365, AI458823, AW02600
3, Y13936, AI380624, AA913312, AI962313, AF04010
5, AI696971, AI264637, AI051599, AF029750, AI98416
6, AI971277, AI792635, X02152, AI286310, AI57183
0, AI928799W28186, T15735, H98135, AI431799, M2341
0, AA564822, AA156784, AA676723, M32578, AA12678
7, AW026119, AI635224, M55914, AI432513, AW00577
5, AI568057, S57501, AI817092, AI742737, AW01593
4, HG1112-HT1112, AI955339, AI986481, AW002819, AB
000095, AA857473, AI816843, AA908890, AI872092, AA
532655, AI743206, AI497609, AF016903, AA034414, AA
677666, AW002409 <Registration number group 2> M25079, AI669685, M69043, AL04829
7, AL050224, AW025584, H05010, AA602984, M12125, A
A497129, L27560, AI749098, Z11793, AI620911, AL046
979, AI983251, X59350, W51743, M96233, AI571452, U
58516, AI566871, AI814178, AI817897, L48215, AI688
582, T53591, AI357209, U77594, AA167094, AI97284
8, AI084193, U15979, AI656836, AI969659, R46698, A
B011170, AA115295, AI982873, R79768, AL050202, AL0
42569, N70077, AL042923, AF035292, N46649, AW01649
6, AA947305, AF037261, N21424, AW008412, AA17681
9, W28281, AI676103, AW003456, AI884831, U83411, H
16294, AW007987, AW001427, M62831, AI983913, H4161
4, AW022852, M99701, AI829724, AI916646, AL03736
3, S68805, AI684486, AI967936, H17681, AB003184, A
W009586, AI828593, AI610837, X77956, N92541, AW006
802, AA194540, L27560, AI146333, AI984087, AI35207
7, X86693, AL043598, AA281145, AI082237, D11428, A
I417267, AW007399, AI968892, M95787, AL043089, AA1
28061, AA143491, M33146, AW022607, AI290237, AI440
424, AL109701, AI769737, AI936699, AL037969, AL050
159, AI807023, R41648, AI983615, AL021154, AA78242
3, AW025487, N99196, X15880, AA194985, N24480, AW0
05790, J03507, AI985704, AA524029, W63545, AB00504
7, AW004042, AI810628, AW007869, AF013570, AW02273
5, AI262287, AI963008, AF052114, AA446219, AF06352
7, AW009564, L13720, R67627, AI075744, AA887130, A
F053944, R60248, AI553918, AW016563, U21128, AA227
065, AA625198, AI620412, M64497, W05842, AL04284
2, AA838843, X52022, W70067, N45224, AW015393, M14
058, AI934584, AI279875, AI922443, AI557295, AA528
255, AA131626, AI208816, X54486, AA503073, AA15016
5, AW007532, X79683, AI031686, AI066756, AW00648
5, U20982, AI858626, AI752406, AA101758, J02854, A
I698134, AI338338, AI004969, U18009, AA906378, AA5
95199, AI979262, U69559, AI125667, AW023627, H4208
5, AL021366, AA146966, AL039400, AI985751, U2239
8, AA521416, AI123567, N50065, M92843, AI016714, A
I564314, AI073544, D78134, AA643513, AI340029, AI8
21126, J04080, AA524436, AL046946, AI300876, AF032
885, AI338536, W73819, AI660901, AB017019, N7813
9, W30943, AI792487, U97105, AI246018, W27481, R54
619, AF049910, AI142548, AI913548, AI333006, AL035
494, AI970823, AI670862, AA897407, U29953, AW05152
7, AA430300, AI376179, W26496, AA523172, AA41138
2, AI418353, AB023209, AI635057, AI928515, AI91743
2, AI651806, AI830095, AI971235, AI022831, Z2255
5, N68231, AA102468, AI092890, X72889, AA778836, A
I860150, AI381576, AA149307, AI982754, AA946755, A
I421663, AI743134, AI887362, W55861, AI557412, AL0
50223, AA479898, AL047415, AA204642, AL046940, AW0
25350, AI688114, AI762319, X13839, AA460952, AA044
844, AI799496, AF055376, AI355848, AI300073, AA147
088, X51345, AI983649, AA526844, AI972597, Y1362
2, AA587245, AI860250, AI890480, AB007898, AW00894
1, AW016298, AI651760, M29039, N25612, AA843523, A
I525812, AL049381, AA557557, N24239, AW021895, V01
512, N66434, AI359214, H67928, D15050, AI990337, A
I810627, AI825981, M62403, AA628405, AI796967, AF0
22813, AI872510, AI052110, AI734967, U17714, AA214
086, AI535730, W52024, AI979070, W63773, AA62147
5, L13720, AI810399, AA531016 2. 2. The method according to the above item 1, wherein the expression level of the gene is measured by measuring the transcript amount of the gene. 3. The method according to item 1 or 2 above, wherein the control value of the expression level of the gene is the value of the expression level of the gene in normal tissues or normal cells In the step (2), (a) the measured value of the expression level of the gene having the nucleotide sequence registered in Genbank with any of the registration numbers shown in the registration number group 1 or its ortholog is higher than the control value. As an indicator,
(B) G in any of the registration numbers shown in the registration number group 2
4. The method according to item 3 above, which detects ovarian cancer cells in a sample, using the measured value of the expression level of a gene having a nucleotide sequence registered in enbank or its ortholog as lower than the control value as an index. A method for evaluating the anticancer activity of a substance, which comprises: (1) contacting an ovarian cancer tissue or ovarian cancer cell with a test substance; (2) tissue contacted with the test substance in the above step (1). Alternatively, a step of measuring the expression level of one or more genes selected from genes having a nucleotide sequence registered in Genbank with any of the accession numbers shown in the above accession numbers 1 or 2 in the cells, or an ortholog thereof, And (3) a step of comparing the measured value of the expression level of the gene obtained in the step (2) with a control value of the expression level of the gene, and evaluating the anticancer activity of the test substance based on the difference. 5. A method characterized by including (hereinafter sometimes referred to as the anticancer activity evaluation method of the present invention), 6. The method according to the above item 5, wherein the expression level of the gene is measured by measuring the transcript amount of the gene. 7. The method according to the above item 5 or 6, wherein the control value of the expression level of the gene is the value of the expression level of the gene in ovarian cancer tissue or ovarian cancer cells. In the step (3), (a) an index is that the measured value of a gene having a nucleotide sequence registered in Genbank with any of the registration numbers shown in the registration number group 1 or its ortholog is lower than the control value, (B) Using the fact that the measured value of the expression level of the gene having the nucleotide sequence registered in Genbank with any of the registration numbers shown in the registration number group 2 or its ortholog is higher than the control value, 8. The method according to the above item 7, which evaluates the anticancer activity; 10. A method for searching an anticancer active substance, which comprises a step of selecting a substance having an anticancer activity based on the anticancer activity evaluated by the method according to any one of the above 5 to 8. 10. An anticancer agent containing, as an active ingredient, an anticancer active substance selected by the search method described in 9 above. A method for evaluating carcinogenic activity of a substance, comprising the steps of (1) contacting an ovary-derived tissue or cell with a test substance, (2) in the tissue or cell contacted with the test substance in the step (1), Registration number group 1 or 2
Measuring the expression level of one or more genes selected from the genes having a nucleotide sequence registered in Genbank with any of the registration numbers shown in 1 or their orthologs, and (3) obtained in step (2) above. The measured value of the expression level of the gene is compared with a control value of the expression level of the gene, and the method comprises the step of evaluating the carcinogenic activity of the test substance based on the difference (hereinafter, the carcinogenic activity of the present invention. It may be referred to as an evaluation method.), 12. 12. The method according to the above item 11, wherein the expression level of the gene is measured by measuring the transcript amount of the gene. 13. The method according to the above item 11 or 12, wherein the control value of the expression level of the gene is the value of the expression level of the gene in normal tissues or normal cells. In the step (3), (a) an index is that the measured value of a gene having a nucleotide sequence registered in Genbank with any of the registration numbers shown in the registration number group 1 or its ortholog is higher than the control value, (B) Genbank with any of the registration numbers shown in the registration number group 2
The carcinogenic activity of the test substance is evaluated by using as an index the measured value of the expression level of the gene having the nucleotide sequence registered in or the ortholog thereof is lower than the control value.
15. The method described in 3 above. A method for searching a carcinogen, comprising a step of selecting a substance having a carcinogenic activity based on the carcinogenic activity evaluated by the method according to any one of the above items 11 to 14.

[0004]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, the gene whose expression level is measured is a gene having a nucleotide sequence registered in Genbank with any of the accession numbers shown in the above accession numbers 1 or 2, or an ortholog thereof. (Hereinafter, it may be collectively referred to as this gene.) One or more genes selected. For information on nucleotide sequences registered in Genbank, see, for example, National Cente
r for Biotechnology Information WEB page (UR
L; http://www.ncbi.nlm.nih.gov) from Genba
The above registration number assigned to each gene by nk
It can be obtained by performing a search based on (Accession No.). The present gene used in the present invention includes, in addition to the gene having the same nucleotide sequence as the above-mentioned known nucleotide sequence, the nucleotide sequence of the above-mentioned gene is different in species of organisms, individual differences or differences between organs and tissues. A gene having a base sequence in which a base is deleted, substituted or added due to a naturally occurring mutation due to the above is also included.

The expression level of this gene can be measured, for example, by
This can be performed by a method of measuring the amount of transcript of the present gene per unit amount of sample, a method of measuring the amount of translation product of the present gene per unit amount of sample, and the like. To measure the amount of transcript of this gene, for example, the amount of mRNA, which is the transcript of the gene, is measured. Specifically, the amount of mRNA of a specific gene can be measured, for example, by quantitative real-time-polymerase chain reaction (hereinafter referred to as quantitative RT-PCR) or Northern hybridization method [J. Samb.
rook, EFFrisch, T. Maniatis; Molecular Cloning 2nd edition,
Cold Spring Harbor Laboratory (Cold S
pring Harbor Laboratory), 1989], DNA array method, in situ hybridization method and the like. To measure the amount of translation product of this gene, for example, the amount of protein encoded by this gene is measured. Specifically, the amount of a specific protein is measured, for example, by an immunological assay method using a specific antibody against the protein (eg, ELISA, Western blot, R
IA, etc.), two-dimensional electrophoresis, high performance liquid chromatography and the like. The specific antibody against the protein encoded by this gene can be prepared by using the protein encoded by this gene as an immunizing antigen according to a conventional method.

The method for measuring the transcript amount of this gene will be further described. The amount of mRNA which is a transcript of this gene is
For example, using a probe or primer designed and prepared based on the nucleotide sequence of the present gene, a general genetic engineering method such as Northern hybridization method, quantitative RT-PCR, DNA array method, in situ high It can be measured by using a hybridization method or the like. Specifically, for example, by J. Sambrook, EFFrisch, T. Maniatis; Molecular Cloning 2nd Edition.
tion), Cold Spring Harbor Laboratory (Cold Spring Harbor Laboratory), 1989 and the like. At this time, a gene whose expression level in tissue is known to be constantly constant (hereinafter referred to as a control gene), for example, β-actin gene (Nucl.Acids.Res., Vol.12, No. .3,
p.1687,1984) and 36B4 (Acidic Ribosomal Phosphoprote
in) (Nucl.Acids.Res., vol.19, No.14, p.3998,1991)
The amount of mRNA such as a gene may be measured at the same time. Then, the expression level of the present gene may be determined by calculating the mRNA amount of the control gene or the amount of the present gene per index value thereof or the index value thereof.

(1 Northern hybridization method)
First, the DNA of the gene whose mRNA amount is to be measured is prepared, and then the DNA consisting of all or part of it is prepared.
Is labeled to prepare a probe. The above gene can be prepared by PCR or the like using a commercially available cDNA (for example, obtained from Takara Shuzo) or a cDNA prepared by the method shown below as a template. For example, first, from the tissue expressing the gene, the guanidine hydrochloride / phenol method, SDS
-Phenol method, guanidine thiocyanate / CsCl
Total RNA is extracted by a usual method such as a method. For example
Total RNA may be extracted using a commercially available kit such as ISOGEN (manufactured by Nippon Gene). For example, mRNA is prepared from the extracted total RNA as follows. First, a poly A column having oligo dT as a ligand was loaded with 5 times or more column volume of Loading buffer [20 mM Tris-HCl buffer (pH 7.6), 0.5 M NaCl, 1 m].
M EDTA, 0.1% (w / v) SDS] is used for equilibration, and then the total RNA prepared by the above method is applied to a column and washed with 10 times column volume of loading buffer.
Furthermore, 5 times column volume of Washing buffer [20 mM Tris-HCl buffer (pH 7.6), 0.1 M NaCl, 1
mM EDTA, 0.1% (w / v) SDS]. Then, 3 times column volume of elution buffer (10 mM Tris-HCl buffer (pH 7.6, 1 mM EDTA, 0.05
MRNA is obtained by eluting the mRNA with% (w / v) SDS). Then, an oligo dT primer is annealed to the poly A chain of the total RNA or mRNA,
For example, single-stranded cDNA is synthesized according to the protocol of cDNA synthesis kit (Takara Shuzo). At this time, the RNA used as a template may be either total RNA or mRNA, but it is more preferable to use mRNA. Using the single-stranded cDNA as a template, TAKARA taq (Takara Shuzo)
DNA by PCR using a DNA polymerase such as
Amplify A. PCR conditions vary depending on the type of animal to be measured, the sequences of the primers used, etc.
For example, reaction buffer [10 mM Tris-HCl buffer (pH 8.
3), 50 mM KCl, 1.5 mM MgCl ₂ ] in the presence of 2.5 mM NTP at 94 ° C. for 30 seconds, then 40 ° C. to 60 ° C. for 2 minutes and 72 ° C. for 2 minutes. Conditions for 55 cycles can be mentioned.

DN of this gene amplified in this way
A may be cloned by inserting it into a vector such as pUC118. The nucleotide sequence of the DNA is Maxam Gilbert
Law (e.g. Maxam, AM & W. Gilbert, Proc.Natl.Acad.Sc
i., 74, 560, 1977) and the Sanger method (for example, Sa
nger, F. & A.R.Coulson, J.Mol.Biol., 94,441,1975, Sang
er, F, & Nicklen and ARCoulson., Proc.Natl.Acad.Sc
i., 74, 5463, 1977, etc.) and the like.

DN of the present gene thus prepared
The probe can be prepared by labeling all or part of A with a radioisotope, a fluorescent dye or the like as follows. For example, using the DNA prepared as described above as a template and an oligonucleotide having a partial sequence of the base sequence of the DNA as a primer, [α-
A probe labeled with ³² P can be obtained by adding dNTP containing ³² P] dCTP or [α- ³² P] dATP to the reaction solution and performing PCR. In addition, DNA prepared as described above
May be labeled using a commercially available labeling kit such as Random prime labeling Kit (Boehringer Mannheim) and MEGALABEL (Takara Shuzo).

Next, Northern hybridization analysis is performed using the above probe. Specifically, total RNA or mRNA is prepared from a tissue or cell whose expression level of this gene is to be measured. Prepared total RNA2
Separate 0 μg or 2 μg of mRNA with agarose gel,
After washing with 10x SSC (1.5M NaCl, 0.35M sodium citrate), a nylon membrane [eg Hybond-N
(Made by Amersham) etc.]. Put the membrane in a polyethylene bag and use the hybridization buffer [6 x S
SC (0.9 M NaCl, 0.21 M sodium citrate), 5 × Denhardt's solution [0.1% (w / v) Ficoll 400, 0.1% (w / v) polyvinylpyrrolidone, 0.1% BSA], 0.1% ( w / v) SDS, 1
00 μg / ml denatured salmon sperm DNA, 50% formamide] 25 ml
After incubating at 50 ° C for 2 hours, the hybridization buffer is discarded, and 2 ml to 6 ml is newly added.
Add ml hybridization buffer. Furthermore, the probe obtained by the above method is added, and the mixture is incubated at 50 ° C. overnight. In addition to the above, commercially available DIG EASY Hyb (Boehringer Mannheim) and the like can be used as the hybridization buffer. Remove the membrane, 50ml-100ml 2xSS
Incubate in C, 0.1% SDS at room temperature for 15 minutes, repeat the same operation once, and finally 50m
Incubate for 30 minutes at 68 ° C. in 1-100 ml of 0.1 × SSC, 0.1% SDS. By measuring the amount of label on the membrane, the amount of mRNA which is a transcription product of this gene can be measured.

(2 Quantitative RT-PCR) mRNA is prepared from a tissue or cell whose expression level is to be measured by a method similar to the method described in (1 Northern hybridization method) above. Prepared mRN
Reverse transcriptase such as MMLV (Toyobo) is added to A, and the reaction buffer [50 mM Tris-HCl buffer (pH 8.
3) 3 mM MgCl ₂ , 75 mM KCl, 10 mM
DTT], 0.5 mM dNTP and 25 μg / m
Reaction is performed at 42 ° C. for 15 minutes to 1 hour in the presence of 1 oligo dT to prepare the corresponding cDNA. The corresponding cDNA may be prepared using a cDNA synthesis kit (Takara Shuzo). PCR using the prepared cDNA as a template and DNA having a part of the base sequence of this gene as a primer
I do. As the primer, for example, Genbank
As a primer having a partial base sequence of the present gene having a base sequence registered under accession number T15777, there can be mentioned a primer comprising the base sequence represented by SEQ ID NO: 1 or 2, and Genbank provides accession number AI3576.
As a primer having a partial nucleotide sequence of this gene having the nucleotide sequence registered in 16, SEQ ID NO: 3 or 4
An example of a primer having a partial base sequence of this gene having a base sequence registered in Genbank under accession number L27560 is a primer consisting of the base sequence shown in SEQ ID NO: 5 or 6. You can give a primer. PCR conditions include, for example, TAKARA taq (Takara Shuzo)
Reaction buffer [10 mM Tris-HCl buffer (p
H 8.3), 50 mM KCl, 1.5 mM MgC
l ₂ ], 2.5 mM dNTP and [α ³² P] -dC
In the presence of TP, for example, 94 ℃, 30 seconds then 40 ℃
Conditions for carrying out this for 30 to 55 cycles can be given, with one cycle being incubation at -60 ° C for 2 minutes and further at 72 ° C for 2 minutes. The amount of mRNA of this gene can be measured by subjecting the amplified DNA to polyacrylamide gel electrophoresis and measuring the amount of radioactivity of the separated DNA. Alternatively, for example, TAKARA taq (Takara Shuzo) is used and a reaction buffer solution [10 mM Tris-HCl buffer solution (pH 8.3), 50 mM KCl, 1.5 mM MgCl ₂ ] is used.
Among them, 50 μL of reaction solution containing 25 μL of SYBR Green PCR Reagents PCR (ABI) was prepared, and after keeping the temperature at 50 ° C. for 2 minutes and then at 95 ° C. for 10 minutes using ABI7700 (ABI), 95 ° C., 15 Insulate for 1 second at 60 ℃ for 1 second
PCR under the condition that 40 cycles are performed as a cycle
I do. By measuring the fluorescence of the amplified DNA,
The amount of mRNA of this gene can be measured.

(3 DNA Array Analysis) To measure the amount of transcript of the present invention, the cDNA of the present gene is spotted on a membrane filter such as a nylon membrane or the like, and the cDNA of the present gene is placed on a slide glass or the like. Microarrays made by spotting, probe arrays made by immobilizing oligonucleotides (usually 18-25mer chain length) having a partial base sequence of this gene on a slide glass using photochemical reaction, etc. A DNA array based on a known technique can be used. These arrays can be prepared according to the method described in, for example, the protocol for research on genomic function, Experimental Medicine Supplement (Yodosha). Alternatively, Genechip or the like commercially available from Affymetrix, Inc. can be used.

An example of a method for measuring the transcript amount of this gene using a DNA array will be shown below. (3-1 Quantification by macroarray) From the tissue or cell whose expression level is to be measured, the above (1
MRNA is prepared by a method similar to the method described in Northern hybridization method). Reverse transcriptase such as MMLV (Toyobo Co., Ltd.) is added to the prepared mRNA, and the reaction buffer [eg, 50 mM Tris-HCl buffer (pH 8.3), 3 mM MgC] is added.
solution containing 1, ₂ , 75 mM KCl, and 10 mM DTT], 0.5 mM d
NTP, [α ³² P] -dCTP, and 25 μg / ml oligo dT
Labeled DNA by reacting in the presence of 42 ° C for 15 minutes to 1 hour
Is prepared and used as a probe. At this time, a cDNA synthesis kit (Takara Shuzo) or the like may be used. A macroarray prepared by spotting the cDNA of this gene on a membrane filter was put in a polyethylene bag, and hybridization buffer [6 x SSC (0.9 M NaCl, 0.21 M sodium citrate), 5 x Denhardt's solution [0.1% (w / v) Ficoll 400, 0.1% (w / v) polyvinylpyrrolidone, 0.1% BS
A], 0.1% (w / v) SDS, 100 μg / ml denatured salmon sperm DNA, 5
After adding 25 ml of 0% formamide] and incubating at 50 ° C. for 2 hours, the hybridization buffer is removed and 2 to 6 ml of hybridization buffer is newly added. Add the above probe and add 50
Incubate at 0 ° C overnight. In addition to the above, commercially available DIG EASY H
yb (Boehringer Mannheim) or the like can also be used. Take out the macro array, 50ml-100ml 2xSS
After soaking in C, 0.1% SDS and incubating for about 15 minutes at room temperature, repeat the same operation once, and finally 50m
Incubate in 1-100 ml of 0.1x SSC, 0.1% SDS at 68 ° C for 30 minutes. By measuring the labeled amount on the macroarray, the amount of mRNA which is a transcript of the present gene, that is, the expression amount of the present gene can be measured.

(3-2 Quantification by Microarray) mRNA is prepared from the tissue or cell whose expression level is to be measured by the same method as described in (1 Northern hybridization method) above. Reverse transcriptase such as MMLV (Toyobo Co., Ltd.) is added to the prepared mRNA, and a reaction buffer [eg, containing 50 mM Tris-HCl buffer (pH 8.3), 3 mM MgCl ₂ , 75 mM KCl, and 10 mM DTT] Liquid], 0.5 mM dNTP, Cy3-dUTP, (or Cy5-dU
TP) and 25 μg / ml oligo dT at 42 ° C. for 15 minutes to 1 hour. Alkaline buffer (eg,
1N NaOH, solution containing 20 mM EDTA) was added and incubated at 65 ° C for 10 minutes, and then free Cy3 or MicroconYM-30 was used.
Fluorescently labeled DNA is prepared by removing Cy5 and used as a probe. Hybridization is performed on the microarray using the obtained probe in the same manner as the method described in (3-1 Quantification by DNA macroarray). By measuring the amount of signal on the array with a scanner, the amount of mRNA that is a transcript of this gene,
That is, the expression level of this gene can be measured.

(3-3 Quantification by probe array) From the tissue or cell whose expression level is to be measured, mRNA is prepared by the same method as described in (1 Northern hybridization method) above. CDNA is prepared from the prepared mRNA using, for example, a cDNA synthesis kit (GENSET). The prepared cDNA can be labeled with, for example, a biotin-labeled cRNA synthesis kit (In Vitro Transcription
(Enzo), etc., labeled with biotin, cRNA clean
up and quantitation kit (In Vitro Transcription
Company). The generated biotin-labeled DNA
Fragmentation buffer (200 mM Tris acetate (pH 8.
1), 500mM KOAc, 150mM MgOAc). Internal standard material Contol Oligo B2 (Amersham),
100 x Control cRNA Cocktail, Herring sperm DNA (Pro
mega), Acetylated BSA (Gibco-BRL), 2 × ME
S Hybridization Buffer 〔200mM MES, 2M [Na ⁺ ], 40mM
EDTA, 0.02% Tween20 (manufactured by Pierce), pH 6.5 to 6.7] and DEPC-treated sterile distilled water are added to prepare a hybrid cocktail. A probe array [eg, Genechip (manufactured by Affymetrix) or the like] filled with 1 × MES hybridization buffer was placed in a hybrid oven at 45 ° C., 60 rpm, 10 rpm.
After spinning for 1 minute, remove 1x MES hybridization buffer. Then, 200 μL of the above-mentioned hybrid cocktail is added to the probe array and rotated in a hybrid oven at 45 ° C., 60 rpm for 16 hours (hybridization). Subsequently, the hybrid cocktail was removed and Non-Stri was removed.
ngent Wash Buffer [6 x SSPE [20 x SSPE (manufactured by Nacalai Tesque)], 0.01% Tween20, and 0.005% Antifoa
m0-30 (including Sigma)] and then GeneChip Fluid
The probe array is mounted in a predetermined position of ics Station 400 (manufactured by Affymetrix) and washed according to the protocol. The probe array is then stained according to the staining protocol EuKGE-WS2 of MicroArray Suite (Affymetrix). 57 by HP GeneArray Scanner (Affymetrix)
By measuring the fluorescence brightness at 0 nm, the amount of mRNA which is a transcript of the present gene, that is, the expression amount of the present gene can be measured.

(4 In Situ Hybridization Method) Basically, it consists of 1) fixing of tissue, embedding, and preparation of section, 2) preparation of probe, and 3) detection by hybridization. Other than using labeled RNA or DNA as a probe, for example, Heiles, H. et al., Biotechnique
s, 6,978,1988, Handbook of genetic engineering, Yodosha 278 19
91, Cell Engineering Handbook, Yodosha, 214, 1992, Cell Engineering Handbook, Yodosha, 222, 1992 and the like. When preparing an RNA probe, first, for example, the DN of the present gene is prepared in the same manner as the method described in (1 Northern hybridization method) above.
A is obtained and the DNA is a vector having SP6, T7 and T3 RNA polymerase promoters (for example, Strata
It is introduced into Escherichia coli by incorporating it into Bluescript manufactured by Gene Co., pGEM manufactured by Promega Co., etc., and plasmid DNA is prepared. Next, the plasmid DNA is cleaved with a restriction enzyme so that sense (for negative control) and antisense (for hybridization) RNA can be produced. These DNA
As a template, in the case of radioactive labeling, α- ³⁵ S-UTP, etc.,
In the case of non-radioactive label, digoxigenin UTP or fluorescein-modified UTP is used as a substrate and SP6, T
7. RNA is synthesized with T3 RNA polymerase while being labeled, and cleaved to a size suitable for hybridization by alkaline hydrolysis to prepare RNA labeled with a radioactive or non-radioactive substance in advance. As a kit based on these methods, for example, an RNA labeling kit (Amersham Co.) for radioactive labeling and DIG RNA for non-radioactive labeling can be used.
Labeling kit (Boehringer Mannheim) or R
NA color kit (Amersham) is commercially available.
When preparing a DNA probe, for example, ³²
Radioactive nucleotide or biotin labeled with P,
Nucleotide labeled with digoxigenin or fluorescein was prepared by the nick translation method (J. Mol. Bio
l., 113,237, Molecular Cloning, A Laboratory Manual 2
nd ed., 10, 6-10, 12, Cold Spring Harbor Lab.) or random prime method (Anal.Biochem., 132,6, Anal.Bioch
em., 137,266), which has been previously labeled with a radioactive or non-radioactive substance.
Prepare A. Kits based on these methods include, for example, Nick Translation Kit (Amersham) and Random Prime Labeling Ki for radioactive labeling.
t (Boehringer Mannheim), but for non-radioactive labeling, DIG DNA labeling kit (Boehringer Mannheim),
A DNA color kit (Amersham) is commercially available. Specifically, the tissue or cells for which the expression level of this gene is to be measured are fixed with paraformaldehyde or the like, embedded in paraffin or the like, and then sliced into thin slices and attached to a slide glass. Alternatively, the above-mentioned tissues or cells are frozen in liquid nitrogen, a microthin section thereof is prepared, and attached to a slide glass. In this way, slide glass sections are obtained. Next, in order to remove substances that react nonspecifically with the probe used in the above tissues or cells, the slide glass sections prepared as described above are treated with proteinase K and acetylated. . Then, the slide glass section is hybridized with the probe prepared as described above. For example, the above probe is heated at 90 ° C. for 3 minutes and then diluted with a hybridization solution, and the solution is dropped on a slide glass section which has been pretreated and covered with a film.
A hybrid is formed by incubating for a time.
After hybridization, non-specific adsorption or unreacted probe is removed by washing or the like (when RNA probe is used, RNase treatment is also added). By measuring the amount of label on the slide glass slice, the amount of mRNA, which is a transcript of this gene, can be measured.

In the detection method of the present invention, the expression level of one or more genes selected from the present gene in a sample is
Measure as described above. When selecting multiple genes whose expression level is to be measured, multiple genes or their orthologs having the nucleotide sequences registered with the accession numbers shown in the same accession number group may be selected, or they may be selected in different accession number groups. A plurality of genes or their orthologs having a nucleotide sequence registered by the registered registration number may be selected.
Among the registration numbers shown in the registration number group 1, genes having the nucleotide sequences registered with the registration numbers shown in the following registration number group 1'or their orthologs are expressed in the ovarian cancer tissue and in the normal tissue. A gene having a larger difference from the level can be preferably mentioned. <Registration number group 1 '> X02344, N37049, U78095, AI98318
1, AB000095, AI147040, AW009505, AW002819, X5734
8, T15777, K03515, AI798743, X74929, AW009649, H98
135, AI497609, AJ131182, AI357616, M55914, AI97933
5 In addition, among the accession numbers shown in accession number group 2, the genes having the nucleotide sequences registered with accession numbers shown in the following accession number group 2'or orthologs thereof have normal expression levels in ovarian cancer tissues and normal levels. A gene having a larger difference from the expression level in a tissue can be preferably mentioned. <Registration number group 2 '> M25079, X86693, AI553918, N9254
1, M14058, M62403, AI300073, AW007532, M12125, M95
787, AI752406, AL043089, X54486, M69043, AI35921
4, AA101758, L48215, AL021154, AL047415, AI03168
6, U20982, L27560, W51743, AI421663, U15979, AF013
570, AA044844, AI698134, U18009, AW007987, W0584
2, AI817897, U83411, U21128, AI810627, AA523172, M
92843, AI290237, AI934584, AW022852, M99701, J0285
4, AI052110, AW025350, J04080, R41648, N78139, AA1
43491, AB003184, U22398, W63773, N66434, U29953, A
I762319, AI566871, AL037969, L27560, D78134, AL046
979, AL042923, AB023209, AL050224, AA115295, W6354
5, M33146, AL046940, AW003456, AW001427, AA14930
7, X77956, U77594, AA887130, AF052114, M29039, AI3
40029, AL037363, X51345, AI557412, M62831, AW01539
3, M64497, V01512, W73819, AI610837, D15050

The sample may be, for example, a biological sample that may contain ovarian cancer tissue or ovarian cancer cells or the contents thereof. Specifically, for example, ovary collected from a test animal. Examples thereof include tissues, ovary cells, oocytes, ovarian germ cells and superficial epithelial cells, tissues such as lymph nodes, body fluids, body secretions and the like. The body fluid can include lymph, blood, plasma, serum and the like, and the body secretion can include urine and the like. These samples may be used as they are,
Further, a sample prepared by various operations such as separation, fractionation, immobilization, etc. from such a sample may be used as a sample. As described above, the expression level of the present gene is measured by the method of measuring the transcript amount of the present gene per unit amount of sample, and the method of measuring the amount of translation product of the present gene per unit amount of sample. And the like, and a method for measuring the transcript amount of the present gene can be preferably mentioned.

The measured value of the expression level of the present gene obtained from the sample as described above is compared with the control value of the expression level of the gene, and ovarian cancer cells in the sample are detected based on the difference. . As a control value of the expression level of the present gene, for example, the expression level value of the gene in normal tissues or normal cells can be mentioned.
Such a control value may be determined by measuring the expression level of the present gene in normal tissues or normal cells in parallel with the expression level of the gene in a sample, or may be determined separately. Alternatively, the expression level of the gene in a plurality of normal tissues or normal cells may be measured and the average value thereof may be used as a control value. For example, using the value of the expression level of this gene in normal tissues or normal cells as a control value, the Ge
If the measured value of the expression level of the gene having the nucleotide sequence registered in nbank or its ortholog in the sample is higher than the control value, it can be assayed that ovarian cancer cells are present in the sample. Further, if the measured value of the expression level of the gene having the nucleotide sequence registered in Genbank with any of the registration numbers shown in the registration number group 2 or its ortholog in the sample is lower than the control value, ovary in the sample is detected. The presence of cancer cells can be assayed.

In the anticancer activity evaluation method of the present invention, first, ovarian cancer tissue or ovarian cancer cells are contacted with a test substance. Examples of the ovarian cancer tissue or ovarian cancer cells that can be used in the anticancer activity evaluation method of the present invention include ovarian cancer tissues or ovarian cancer cells derived from mammals. Examples of ovarian cancer cells include cell lines prepared from ovarian cancer tissue, primary culture cells, and the like. Ovarian cancer cells can be prepared, for example, as follows. First, an ovarian cancer tissue is extracted, shredded, and then digested with an enzyme such as collagenase to obtain a cell suspension. This is Shillabeer G. et al., Internatio
According to the method described in nal Journal of Obesity 20, S77-S83, etc., a fraction rich in ovarian cancer cells can be prepared by fractionating by centrifugation and collecting the precipitate. Culturing is carried out by a conventional method (for example, bio-experiment illustrated quickly, cell culture, 19
1996, published by Shujunsha, etc.). For example, it is cultured at 37 ° C. in the presence of 5% CO ₂ .

When the ovarian cancer tissue or ovarian cancer cells are brought into contact with the test substance in the anticancer activity evaluation method of the present invention, the concentration of the test substance is about 0.1 μM to about 100.
It can be increased to about μM, usually about 1 μM to about 50 μ
About M is preferable. The time for contacting the ovarian cancer tissue or ovarian cancer cells with the test substance is, for example, about 1 hour to
It is about 5 days, and usually several hours to about 2 days is preferable. The culture conditions during the contact may be around 37 ° C. in the presence of 5% CO ₂ . For contacting the test substance with ovarian cancer tissue or ovarian cancer cells, for example, in a solution containing a salt for buffer such as sodium phosphate in water, a medium component usually used when culturing animal cells was contained. It is performed in a solution or the like.

Next, the expression level of one or more genes selected from the present gene in the ovarian cancer tissue or ovarian cancer cells contacted with the test substance as described above is measured as described above. When selecting multiple genes whose expression level is to be measured, multiple genes or their orthologs having the nucleotide sequences registered with the accession numbers shown in the same accession number group may be selected, or they may be selected in different accession number groups. A plurality of genes or their orthologs having a nucleotide sequence registered by the registered registration number may be selected. A gene having a nucleotide sequence registered with the registration number shown in the above registration number group 1'or 2'or an ortholog thereof is a gene having a larger difference between the expression level in ovarian cancer tissue and the expression level in normal tissue. Can be preferably mentioned as As described above, the expression level of the present gene is measured by the method of measuring the transcript amount of the present gene per unit amount of sample, and the method of measuring the amount of translation product of the present gene per unit amount of sample. And the like, and a method for measuring the transcript amount of the present gene can be preferably mentioned.

The measured value of the expression level of the present gene obtained from the ovarian cancer tissue or ovarian cancer cell contacted with the test substance as described above is compared with the control value of the expression level of the gene, The anticancer activity of the test substance is evaluated based on the difference. Examples of the control value of the expression level of the present gene include the expression level of the gene in ovarian cancer tissue or ovarian cancer cells that have not been contacted with the test substance. The control value is the expression level of the gene in ovarian cancer tissue or ovarian cancer cells not contacted with the test substance, and the expression level of the gene in ovarian cancer tissue or ovarian cancer cells contacted with the test substance. The measurement may be performed in parallel with the above, or the measurement may be performed separately. For example, a part of ovarian cancer tissue or ovarian cancer cells before contact with a test substance may be collected to measure the expression level of this gene, and the obtained value may be used as a control value. Alternatively, the expression level of the gene in a plurality of ovarian cancer tissues or ovarian cancer cells may be measured and the average value thereof may be used as a control value. For example, a gene having a nucleotide sequence registered in Genbank with any of the accession numbers shown in Accession No. 1 using the value of the expression level of this gene in ovarian cancer tissue or ovarian cancer cells as a control value, or If the measured value of the expression level of the ortholog in the ovarian cancer tissue or ovarian cancer cells contacted with the test substance is lower than the above control value, the ovarian cancer tissue or ovarian cancer cells It means a decrease, and the test substance can be evaluated as having anticancer activity. In addition, the expression level of the gene having the nucleotide sequence registered in Genbank with any of the registration numbers shown in the registration number group 2 or an ortholog thereof in the ovarian cancer tissue or ovarian cancer cells contacted with the test substance If the measured value is higher than the control value, it means that the ovarian cancer tissue or ovarian cancer cells is decreased by contact with the test substance, and the test substance can be evaluated as having anticancer activity.

By administering a test substance to an animal having ovarian cancer tissue or ovarian cancer cells, the tissue or ovarian cancer cells present in the body of the animal and the test substance are brought into contact with each other to prevent By carrying out the cancer activity evaluation method, the anticancer activity of the test substance at the individual level of the animal can also be evaluated. Examples of the animal include a natural animal, a transgenic animal, a gene knockout animal, an animal transplanted with an ovarian cancer tissue, and the like. For example, it has a nucleotide sequence registered in Genbank with any of the registration numbers shown in the registration number group 1 with the value of the expression level in the ovarian cancer tissue or ovarian cancer cells before administration of the test substance as a control value. If the measured value of the expression level of the gene or its ortholog in the ovarian cancer tissue or ovarian cancer cells contacted with the test substance is lower than the control value, it is evaluated that the test substance has anticancer activity. be able to. Also, with any of the registration numbers shown in the registration number group 2,
If the measured value of the expression level of the gene having the nucleotide sequence registered in k or its ortholog in the ovarian cancer tissue or ovarian cancer cell contacted with the test substance is higher than the control value, the test substance Can be evaluated to have anti-cancer activity.

An anticancer active substance can be searched for by selecting a substance having an anticancer activity based on the anticancer activity evaluated by the anticancer activity evaluation method of the present invention. The selected anticancer active substance can be used as an active ingredient of an anticancer agent.

The anticancer active substance may be used as an active ingredient of an anticancer agent in the form of a pharmaceutically acceptable acid addition salt or base addition salt of the substance. Examples of acid addition salts include inorganic acid addition salts and organic acid addition salts, examples of inorganic acid addition salts include hydrochlorides, hydrobromides, sulfates, phosphates, and the like, and organic acid addition salts include acetates. , Methanesulfonate, benzenesulfonate, citrate, fumarate,
Maleate, succinate, tartrate, malate and the like can be mentioned. Examples of the base addition salt include an inorganic base addition salt or an organic base addition salt, and as the inorganic base addition salt, sodium salts, alkali metal salts such as potassium salts, magnesium salts, alkaline earth metal salts such as calcium salts,
Examples thereof include ammonium salts, and examples of the organic base addition salt include basic amino acid addition salts such as arginine salt and lysine salt. Such salt can be produced by a known means. In addition, the anticancer active substance may be used as an active ingredient of the anticancer agent in the form of a solvate such as a hydrate of a pharmaceutically acceptable salt of the substance.

Administration of the above-mentioned anticancer agent is carried out by a usual administration route,
For example, it can be performed orally, intramuscularly, intravenously, subcutaneously, intraperitoneally, intranasally and the like. The dose and the number of administrations vary depending on the administration route, the degree of symptoms, age, body weight and the like, but usually about 1 mg to 1 g per day for an adult is administered once or more times a day. As the dosage form,
Examples thereof include powders, fine granules, granules, tablets, capsules, suppositories, injections and nasal preparations. In the case of formulation, it can be produced by a conventional method using a usual formulation carrier. That is, when preparing an oral preparation, after adding a binder, a disintegrating agent, a lubricant, a coloring agent, etc., if necessary,
Tablets, granules, powders, capsules and the like are prepared by a conventional method.
When preparing an injection, a pH adjusting agent, a buffer, a stabilizer, a solubilizing agent, etc. are added if necessary, and the injection is prepared by a conventional method.

In the method for evaluating carcinogenic activity of the present invention, first, a tissue having a possibility of developing ovarian cancer, specifically, for example, a tissue or cell derived from ovary is brought into contact with a test substance. The ovary-derived tissue or cells that can be used in the method for evaluating carcinogenic activity of the present invention include ovarian tissue-derived cells or cells derived from mammals,
Examples thereof include ovary cells, oocytes, ovarian germ cells and superficial epithelial cells. Examples of the cells include cell lines, primary culture cells and the like. Culturing of these tissues or cells can be carried out according to a usual method (for example, a method described in Bio Experiment Illustrated Sukusuku Grow Cell Culture, 1996, published by Shujunsha). For example, it is cultured at 37 ° C. in the presence of 5% CO ₂ .

When the test substance is brought into contact with the tissue or cells derived from the ovary in the method for evaluating carcinogenic activity of the present invention, the concentration of the test substance can be about 0.1 μM to about 100 μM, usually about 1 μM to About 50 μM is preferable. The time for contacting the test substance with the ovarian cancer tissue or cells is, for example, about 1 hour to about 5 days, and usually several hours to about 2 days are preferable.
The culture conditions during the contact are about 37 ° C. and 5%.
The presence of CO ₂ can be mentioned. Contact between the test substance and tissues or cells derived from the ovary can be performed, for example, in water, in a solution containing a salt for buffer such as sodium phosphate, in a solution containing medium components usually used when culturing animal cells, etc. Done in.

Then, the expression level of one or more genes selected from the present gene in the ovary-derived tissue or cells contacted with the test substance as described above is measured as described above. When selecting multiple genes whose expression level is to be measured, multiple genes or their orthologs having the nucleotide sequences registered with the accession numbers shown in the same accession number group may be selected, or they may be selected in different accession number groups. A plurality of genes or their orthologs having a nucleotide sequence registered by the registered registration number may be selected. The gene having the nucleotide sequence registered with the registration number shown in the above registration number group 1'or 2'or an ortholog thereof is a gene having a large difference between the expression level in ovarian cancer tissue and the expression level in normal tissue. Can be preferably mentioned as To measure the expression level of this gene,
As described above, it can be carried out by a method of measuring the amount of transcript of the present gene per unit amount of sample, a method of measuring the amount of translation product of the present gene per unit amount of sample, etc. A preferable method is to measure the amount of the transcript.

The measured value of the expression level of the gene obtained from the ovary-derived tissue or cell contacted with the test substance as described above is compared with the control value of the expression level of the gene, and based on the difference. Evaluate the carcinogenic activity of the test substance. As a control value of the expression level of the present gene, for example, the expression level value of the gene in normal tissues or normal cells can be mentioned. Such a control value is
The expression level of this gene in normal tissues or cells may be measured in parallel with the expression level of the gene in ovary-derived tissues or cells contacted with the test substance, or may be determined separately. Good. For example,
It is also possible to collect a part of ovary-derived tissue or cells before contacting with the test substance, measure the expression level of this gene, and use the obtained value as a control value. Alternatively, the expression level of the gene in a plurality of normal tissues or normal cells may be measured and the average value thereof may be used as a control value. For example,
Using a value of the expression level of this gene in normal tissues or normal cells as a control value, a test substance of a gene having a nucleotide sequence registered in Genbank with any of the accession numbers shown in accession number group 1 or its ortholog If the measured value of the expression level in the contacted ovary-derived tissue or cell is higher than the control value, it means the development of ovarian cancer tissue or ovarian cancer cell by contact with the test substance, and the test substance is It can be evaluated to have carcinogenic activity. In addition, the measured value of the expression level of the gene having the nucleotide sequence registered in Genbank with any of the registration numbers shown in the registration number group 2 or its ortholog in the tissue or cells derived from the ovary contacted with the test substance is If it is lower than the control value, it means the development of ovarian cancer tissue or ovarian cancer cells by contact with a test substance, and the test substance can be evaluated as having carcinogenic activity.

Carcinogenesis of the test substance at the individual level of the animal by administering the test substance to an animal, bringing the tissue or cells derived from the ovary of the animal into contact with the test substance, and carrying out the method for evaluating carcinogenic activity of the present invention. Activity can also be assessed. Examples of animals include natural animals, transgenic animals, gene knockout animals, and the like. For example, using a value of the expression level in normal tissue or normal cells as a control value, a gene having a nucleotide sequence registered in Genbank with any of the accession numbers shown in accession number group 1 or its ortholog is contacted with a test substance. If the measured value of the expression level in the tissue or cells derived from the ovary is higher than the control value, it can be evaluated that the test substance has carcinogenic activity. In addition, the measured value of the expression level of the gene having the nucleotide sequence registered in Genbank with any of the registration numbers shown in the registration number group 2 or its ortholog in the tissue or cells derived from the ovary contacted with the test substance is If it is lower than the control value, it can be evaluated that the test substance has carcinogenic activity.

The carcinogenic activity evaluation method of the present invention as described above can be used for detection of substances having carcinogenic activity. Further, a carcinogenic active substance can be searched for by selecting a substance having a carcinogenic activity based on the carcinogenic activity evaluated by the carcinogenic activity evaluation method of the present invention. The selected carcinogenic active substance can also be used, for example, for producing an ovarian cancer model animal.

[0034]

The present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

Example 1 Measurement of expression level of this gene Preparation of total RNA 10 ml of TRIZOL Reagent (manufactured by Gibco-BRL) was added to 1 g wet weight of human ovarian cancer tissue and normal tissue, respectively, and polytron was cooled with ice. Homogenize with a homogenizer and leave for 5 minutes at room temperature. Then 4 ℃, 9,000rpm,
After centrifuging for 10 minutes, the aqueous layer is separated into a 50 ml centrifuge tube (IW
AKI). Add 1/5 volume of TRIZOL Reagent chloroform (Kanto Chemical Co., Inc.), stir vigorously up and down for 15 seconds, and then leave at room temperature for 5 minutes. Then, 4 ℃, 9,
After centrifugation at 000 rpm for 10 minutes, the aqueous layer is collected in a new 50 ml centrifuge tube. Further, 1/2 volume of TRIZOL Reagent 2-propanol (manufactured by Kanto Chemical Co., Inc.) is added, mixed by inversion, and then allowed to stand at room temperature for 10 minutes. After centrifugation at 4 ° C and 9,000 rpm for 10 minutes, the supernatant is removed to obtain a pellet. The obtained pellet is dissolved with 1/10 volume of TRIZOL Reagent, DEPC-treated sterile distilled water (manufactured by Wako Pure Chemical Industries, Ltd.). Add 0.1 ml of the obtained RNA solution to the RLT buff attached to the RNeasy Mini Kit (QIAGEN)
er (10 μL β-mercaptoethanol / mL RLT buffer) 3
Add 50 μL and mix. In addition, 250 μL ethanol
(Kanto Chemical Co., Inc.) is added and mixed. RNeasy the mixture
Apply to the RNeasy Spin Column attached to the Mini Kit and centrifuge at room temperature, 10,000 rpm for 15 seconds. After centrifugation, apply the eluate again to the same RNeasy Spin Column and centrifuge at room temperature, 10,000 rpm for 15 seconds. After centrifugation, discard the eluate and dilute 4 times with ethanol RNeasy Mini Kit
Apply 500 μL of the attached RPE buffer, room temperature, 10,000 rp
Centrifuge for 15 seconds at m. After centrifugation, discard the eluate,
Apply 500 μL of RPE buffer diluted with ethanol,
Centrifuge at room temperature, 14,000 rpm for 2 minutes. Then transfer the column to a new Eppendorf tube and transfer it to the RNeasy Mini.
Add 50 μL of distilled water supplied with the Kit and let stand for 1 minute at room temperature. After standing, elute by centrifugation at room temperature at 10,000 rpm for 1 minute to obtain total RNA. For normal tissues, perform the same procedure as above to prepare total RNA.

Total RNA prepared from ovarian cancer tissue or total RNA prepared from normal tissue in cDNA preparation, 10 μg, T7- (dT) 24 primer
(Amersham) 11 μL of the mixed solution containing 100 pmol was added to 70
After heating at ℃ for 10 minutes, cool on ice. After cooling, SuperScr
ipt Choice System for cDNA Synthesis (Gibco-BRL)
4 μL of 5 × First Strand cDNA Buffer contained in the kit, 2 μL of 0.1 M DTT contained in the kit, and 1 μL of 10 mM dNTPMix contained in the kit are added, and the mixture is heated at 42 ° C. for 2 minutes.
In addition, 2 μL (400 μL) of Super ScriptII RT included in the kit
U) is added, and the mixture is heated at 42 ° C for 1 hour and then cooled on ice. After cooling, 91 μL of sterile distilled water treated with DEPC, included in the kit 5
× Second Strand Reaction Buffer 30 μL, 10 mM dNTP M
ix 3 μL, E. coli DNA Ligase 1 μL included in the kit
(10U), E. coli DNA Polymerase I included in the kit
4 μL (40 U), and E. coli RNaseH included in the kit
Add 1 μL (2U) and incubate at 16 ℃ for 2 hours. Then
After adding 2 μL (10 U) of T4 DNA Polymerase contained in the kit and reacting at 16 ° C. for 5 minutes, 10 μL of 0.5 M EDTA is added. Then, 162 μL of phenol / chloroform / isoamyl alcohol solution (manufactured by Nippon Gene Co., Ltd.) was added,
Mix. The mixture was transferred to Phase Lock Gel Light (manufactured by Eppendorf Co.) that had been centrifuged at room temperature, 14,000 rpm for 30 seconds in advance, and the mixture was centrifuged at room temperature, 14,000 rpm for 2 minutes, and then 1
Transfer 45 μL of the aqueous layer to an Eppendorf tube. Add 72.5 μL of 7.5 M ammonium acetate solution and 36
Add 2.5 μL and mix, then centrifuge at 4 ° C, 14,000 rpm for 20 minutes. After centrifugation, the supernatant is discarded and a DNA pellet is obtained. Then, add 0.5 mL of 80% ethanol to the DNA pellet, centrifuge at 4 ° C, 14,000 rpm for 5 minutes, and discard the supernatant. Add again 0.5 mL of 80% ethanol to the DNA pellet and
After centrifuging at 14,000 rpm for 5 minutes at ℃, after discarding the supernatant, the pellet is dried and dissolved in 12 μL of sterile distilled water treated with DEPC to obtain a cDNA solution. On the other hand, to
For tal RNA, a cDNA solution is prepared by performing the same operation as above.

Preparation of Labeled cRNA Labeled cRNAs are prepared using cDNA derived from ovarian cancer tissue and cDNA derived from normal tissue, respectively. That is,
5 μL of the cDNA solution obtained in step 1, 17 μL of DEPC-treated sterile distilled water
L, BioArray High Yield RNA Transcript Labeling Kit
(ENZO) 10 × HY Reaction Buffer 4 μL,
10x Biotin Labeled Ribonucleotid included in the kit
es 4 μL, 10 × DTT 4 μL included in the kit, 10 × RNase Inhibitor Mix 4 μL included in the kit, and 20 × T7 RNA Polymerase 2 μL included in the kit are mixed,
Incubate at 37 ℃ for 5 hours. After adding 60 μL of sterile distilled water treated with DEPC to the reaction solution, the RNeasy Mini Ki described in
Purify the labeled cRNA using t.

Fragmentation of labeled cRNA Purified labeled cRNA 20 μg, 5 × Fragmentation Buf
fer (200 mM Tris-acetic acid pH8.1 (Sigma), 500 mM potassium acetate (Sigma), and 150 mM magnesium acetate
(Manufactured by Sigma)) 40 μL of a reaction solution containing 8 μL was heated at 94 ° C. for 35 minutes and then cooled with ice to obtain a fragmented cRNA solution.

Hybridization of Fragmented cRNA with Probe Array Fragmented cRNA derived from ovarian cancer tissue and fragmented cRNA derived from normal tissue are each hybridized with a probe array as follows. That is,
Fragmented cRNA solution obtained in step 40 μL, 5 nM Cont
ol Oligo B2 (Amersham) 4 μL, 100 x Control cRN
A Cocktail 4 μL, Herring sperm DNA (Promega) 4
0 μg, Acetylated BSA (Gibco-BRL) 200 μg, 2 × ME
S Hybridization Buffer (200mM MES, 2M [Na ⁺ ], 40mM E
DTA, 0.02% Tween20 (manufactured by Pierce), pH 6.5 to 6.7) 200μ
400 μL by mixing L and 144 μL of sterile distilled water treated with DEPC
To obtain a hybrid cocktail of. The obtained hybrid cocktail is heated at 99 ° C for 5 minutes, and further at 45 ° C for 5 minutes. After heating, centrifugation is performed at room temperature and 14,000 rpm for 5 minutes to obtain a hybrid cocktail supernatant. Human Genome U95 Probe Array (Affymet) filled with 1x MES hybridization buffer
rix) in a hybrid oven at 45 ° C, 60 rpm, 10 rpm
After spinning for 1 minute, remove 1x MES hybridization buffer. Then, 200 μL of the hybrid cocktail supernatant is added to the probe array and rotated in a hybrid oven at 45 ° C. at 60 rpm for 16 hours to obtain a hybridized probe array.

The hybrid cocktail was removed from the hybridized probe array obtained by staining the probe array, and the non-stringent wash buffer was used.
[6 × SSPE (diluted 20 × SSPE (manufactured by Nacalai Tesque)), 0.0
Including 1% Tween20]. GeneChip Fluidics Stati
on 400 (manufactured by Affymetrix Co.) was mounted the probe array in a predetermined position, after washing the probe array according to the protocol, the probe array according to the following method according to the staining protocol EuKGE-WS2 of MicroArray Suite (Affymetrix Co.) To dye. First, the primary staining solution [10 μg /
mL Streptavidin Phycoerythrin (SAPE) (Molecular Pro
be manufactured), 2 mg / mL Acetylated BSA, 100 mM MES, 1 M NaCl
(Made by Ambion) containing 0.05% Tween 20] for 45 minutes. Next, secondary staining solution [100 μg / mL Goat IgG (Sigma), 3 μg / mL Biotinylated Anti-Streptavidinantibod
y (Vector Laboratories), 2mg / mL Acetylated BS
A, 100 mM MES, 1M NaCl, 0.05% Tween20, and 0.005% A
Includes ntifoam0-30] for 10 minutes, and finally for 45 minutes in the primary dyeing solution.

The probe array derived from the ovarian cancer tissue and the probe array derived from the normal tissue, which were stained by scanning and analysis of the probe array, were respectively used as HP GeneArray.
Use a scanner (manufactured by Affymetrix) to measure the signal at 570 nm
Read by measuring the fluorescence intensity of. The obtained results were compared and analyzed by GeneChip Microarray Suite (manufactured by Affymetrix), and genes having different expression levels in normal tissue and ovarian cancer tissue were extracted. In the gene having a nucleotide sequence registered in Genbank with any of the registration numbers shown in the registration number group 1, the signal detected in the probe array derived from the above ovarian cancer tissue is The detected signal was exceeded. <Registration number group 1> X02344, AI884621, X59932, AI98943
2, X74929, AI346658, AI354351, AI332641, U28964, A
I672414, D26598, H44597, U65785, AA775776, AA74243
8, AI887311, M13560, AI807277, D38047, AI798743, X
04366, AI791182, H15313, AA004221, L24521, AI95497
5, D45248, AI973186, AF051782, AI762891, N37049, A
A554681, X57346, W21894, U48705, AA112121, AL05029
0, AW024537, W60640, AW024572, M26252, AA020846, U
85611, R54450, U78095, AI983181, AI741591, AI99112
2, W28869, AI147040, HG1614-HT1614, AI986430, AJ00
2308, AA062899, AA532392, AI560151, Y15915, AI3010
60, HG1980-HT2023, AI351790, M21186, AL041566, AI8
31655, AA211690, AI885170, AI241578, M91670, AW020
965, D42123, N32858, AI460118, AI539386, AI62038
1, AI804075, U21689, R20554, D89052, AI684451, AA4
79496, AA131307, X57348, AI992284, U77604, AI66041
2, AF054174, AI878836, AI991845, AW001374, AF00539
2, AI862775, U47634, AA928646, M83751, AI341234, A
I018296, AI394307, Z69043, AI032386, X00734, AI970
195, U49278, AI741313, AI634570, AI733988, AJ13118
2, AI613214, X67325, AW007442, AI653621, AA27853
8, T15777, AA543084, J03909, AA587236, L16842, AI5
57649, M34423, AI309320, AA622029, AI934540, Z9393
0, AA383718, X69699, AI979335, M65028, AA044793, A
I357616, AI417084, AF047436, AW009649, AW004750, A
I990317, AW007731, AI087923, AI867409, AI660570, A
F001601, N63815, AA004274, AI927408, K03515, AI624
028, AA576813, AI627731, M61832, AI923108, AW00950
5, AI969418, X73478, AI589365, AI458823, AW02600
3, Y13936, AI380624, AA913312, AI962313, AF04010
5, AI696971, AI264637, AI051599, AF029750, AI98416
6, AI971277, AI792635, X02152, AI286310, AI57183
0, AI928799, W28186, T15735, H98135, AI431799, M23
410, AA564822, AA156784, AA676723, M32578, AA12678
7, AW026119, AI635224, M55914, AI432513, AW00577
5, AI568057, S57501, AI817092, AI742737, AW01593
4, HG1112-HT1112, AI955339, AI986481, AW002819, AB
000095, AA857473, AI816843, AA908890, AI872092, AA
532655, AI743206, AI497609, AF016903, AA034414, AA
677666, AW002409

On the other hand, in the case of a gene having a nucleotide sequence registered in Genbank with any of the accession numbers shown in accession number group 2, the signal detected by the above-mentioned probe array derived from normal tissue is ovarian cancer tissue. It exceeded the signal detected in the probe array of origin. <Registration number group 2> M25079, AI669685, M69043, AL04829
7, AL050224, AW025584, H05010, AA602984, M12125, A
A497129, L27560, AI749098, Z11793, AI620911, AL046
979, AI983251, X59350, W51743, M96233, AI571452, U
58516, AI566871, AI814178, AI817897, L48215, AI688
582, T53591, AI357209, U77594, AA167094, AI97284
8, AI084193, U15979, AI656836, AI969659, R46698, A
B011170, AA115295, AI982873, R79768, AL050202, AL0
42569, N70077, AL042923, AF035292, N46649, AW01649
6, AA947305, AF037261, N21424, AW008412, AA17681
9, W28281, AI676103, AW003456, AI884831, U83411, H
16294, AW007987, AW001427, M62831, AI983913, H4161
4, AW022852, M99701, AI829724, AI916646, AL03736
3, S68805, AI684486, AI967936, H17681, AB003184, A
W009586, AI828593, AI610837, X77956, N92541, AW006
802, AA194540, L27560, AI146333, AI984087, AI35207
7, X86693, AL043598, AA281145, AI082237, D11428, A
I417267, AW007399, AI968892, M95787, AL043089, AA1
28061, AA143491, M33146, AW022607, AI290237, AI440
424, AL109701, AI769737, AI936699, AL037969, AL050
159, AI807023, R41648, AI983615, AL021154, AA78242
3, AW025487, N99196, X15880, AA194985, N24480, AW0
05790, J03507, AI985704, AA524029, W63545, AB00504
7, AW004042, AI810628, AW007869, AF013570, AW02273
5, AI262287, AI963008, AF052114, AA446219, AF06352
7, AW009564, L13720, R67627, AI075744, AA887130, A
F053944, R60248, AI553918, AW016563, U21128, AA227
065, AA625198, AI620412, M64497, W05842, AL04284
2, AA838843, X52022, W70067, N45224, AW015393, M14
058, AI934584, AI279875, AI922443, AI557295, AA528
255, AA131626, AI208816, X54486, AA503073, AA15016
5, AW007532, X79683, AI031686, AI066756, AW00648
5, U20982, AI858626, AI752406, AA101758, J02854, A
I698134, AI338338, AI004969, U18009, AA906378, AA5
95199, AI979262, U69559, AI125667, AW023627, H4208
5, AL021366, AA146966, AL039400, AI985751, U2239
8, AA521416, AI123567, N50065, M92843, AI016714, A
I564314, AI073544, D78134, AA643513, AI340029, AI8
21126, J04080, AA524436, AL046946, AI300876, AF032
885, AI338536, W73819, AI660901, AB017019, N7813
9, W30943, AI792487, U97105, AI246018, W27481, R54
619, AF049910, AI142548, AI913548, AI333006, AL035
494, AI970823, AI670862, AA897407, U29953, AW05152
7, AA430300, AI376179, W26496, AA523172, AA41138
2, AI418353, AB023209, AI635057, AI928515, AI91743
2, AI651806, AI830095, AI971235, AI022831, Z2255
5, N68231, AA102468, AI092890, X72889, AA778836, A
I860150, AI381576, AA149307, AI982754, AA946755, A
I421663, AI743134, AI887362, W55861, AI557412, AL0
50223, AA479898, AL047415, AA204642, AL046940, AW0
25350, AI688114, AI762319, X13839, AA460952, AA044
844, AI799496, AF055376, AI355848, AI300073, AA147
088, X51345, AI983649, AA526844, AI972597, Y1362
2, AA587245, AI860250, AI890480, AB007898, AW00894
1, AW016298, AI651760, M29039, N25612, AA843523, A
I525812, AL049381, AA557557, N24239, AW021895, V01
512, N66434, AI359214, H67928, D15050, AI990337, A
I810627, AI825981, M62403, AA628405, AI796967, AF0
22813, AI872510, AI052110, AI734967, U17714, AA214
086, AI535730, W52024, AI979070, W63773, AA62147
5, L13720, AI810399, AA531016

Example 2 Detection of Ovarian Cancer Cells Using Probe Array Total RNA was prepared from two or more different biopsy samples including a biopsy sample derived from a healthy person by the same procedure as in Example 1. After that, a fragmented cRNA derived from a biopsy sample derived from a healthy person and a fragmented cRNA derived from a test biopsy sample are prepared by the same operations as in Example 1 to.
Next, by the same operation as in Example 1, a fragmented cRNA derived from a biopsy sample derived from a healthy person and a fragmented cRNA derived from a test biopsy sample were converted into an oligonucleotide having a partial base sequence of the base sequence of this gene. Stain after hybridizing to the immobilized probe array.
The stained probe array obtained was used for HP GeneArray Sca.
The signal is read by measuring the fluorescence intensity at 570 nm, and the obtained results are compared and analyzed by GeneChip Microarray Suite (Affymetrix). The expression level of a gene having a nucleotide sequence registered in Genbank with any of the accession numbers shown in accession numbers 1 or 2 or its ortholog was compared between a biopsy sample from a healthy subject and a biopsy sample of a subject. Detect ovarian cancer cells in the test biopsy sample based on the difference. That is, the measured value of the expression level in the test biopsy sample of the gene or its ortholog having the nucleotide sequence registered in Genbank with any of the registration numbers shown in the registration number group 1 is in the biopsy sample derived from a healthy person. If the expression level of the gene is higher, it is assayed that ovarian cancer cells are present in the test biopsy sample. Also, if any of the registration numbers shown in the registration number group 2
If the measured value of the expression level of the gene having the nucleotide sequence registered in nbank or its ortholog in the test biopsy sample is lower than the expression level of the gene in the biopsy sample derived from a healthy person, the test biopsy sample Assay for the presence of ovarian cancer cells in it.

Example 3 Detection of Ovarian Cancer Cells Using Quantitative RT-PCR Total RNA was prepared from two or more different biopsy samples including a biopsy sample derived from a healthy person by the same procedure as in Example 1. Then, the cDNA from the biopsy sample derived from a healthy subject and the c from the test biopsy sample were prepared by the same procedure as in Example 1.
Prepare DNA. Next, using the prepared cDNA as a template,
PCR is carried out as follows, and the amplified DNA is quantified. That is, 1 μL of the cDNA, 20 pmol of the primer 1 consisting of the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 2
Includes 20 pmol of primer 2 consisting of the nucleotide sequence shown in 5 and 25 μL of SYBR Green PCR Reagents PCR (ABI) 5
Prepare 0 μL of reaction solution and use ABI7700 (ABI) at 50 ℃
After incubating for 2 minutes and then 95 ℃ for 10 minutes, 95 ℃ for 15 seconds and then 60
PCR is carried out under the condition that 40 cycles of this is performed by keeping 1 minute of incubation at 1 ° C. From the amount of amplified DNA,
The expression level of the gene in the test biopsy sample and the expression level of the gene in the biopsy sample derived from a healthy person are determined. Genbank with any of the registration numbers shown in registration number group 1
If the measured value of the expression level of the gene having the nucleotide sequence registered in or the ortholog thereof in the test biopsy sample is higher than the expression level of the gene in the biopsy sample derived from a healthy person, in the test biopsy sample Test for the presence of ovarian cancer cells in. Moreover, the measured value of the expression level in the test biopsy sample of the gene having the nucleotide sequence registered in Genbank with any of the registration numbers shown in the registration number group 2 or the ortholog thereof in the biopsy sample derived from a healthy person is If the expression level of the gene is lower, the presence of ovarian cancer cells in the test biopsy sample is assayed.

Example 4 Preparation of Anti-Cancer Activity Evaluation Cell Using Probe Array 3.0 × 10 ⁵ cells of 6-well plate (Sumitomo Bakelite Co., Ltd.) were seeded with ovarian cancer-derived cultured cells. F
Culture using a BS-containing medium at 37 ° C. in the presence of 5% carbon dioxide for 2 nights. Next, the medium is exchanged with an FBS-containing medium to which a solution prepared by dissolving the test substance in 0.1% DMSO is added, and then the medium is cultured for 2 hours or 48 hours to obtain a cell culture solution of the test substance-added section. On the other hand, a cell culture solution containing no test substance is prepared by culturing in the same manner as above except that 0.1% DMSO is used instead of the test substance solution.

Gene Expression Analysis From the cells from the test substance-added group and the cells from the non-added group, the total amount was calculated by the same procedure as in
After RNA is prepared, fragmented cRNA derived from cells in each of the test compound-added group and the non-added group is prepared by the same operations as in Example 1 to. Next, by the same operation as in Example 1, the fragmented cRNAs derived from the cells in the test substance-added group and the non-added group were applied to a probe array on which an oligonucleotide having a partial base sequence of the base sequence of the present gene was immobilized. After hybridizing, stain. The HP Gene Array Scanner (Aff
ymetrix) and read the signal by measuring the fluorescence intensity at 570 nm, and obtain the obtained results with GeneCh
Comparative analysis is performed using ip Microarray Suite (manufactured by Affymetrix). The expression level of the gene having the nucleotide sequence registered in Genbank with any of the registration numbers shown in the registration number group 12 or its ortholog in the test substance-added group is 1/2 of the expression level in the test substance-free group.
The test substance is evaluated to have anti-cancer activity if it is below. In addition, the expression level of the gene having the nucleotide sequence registered in Genbank with any of the registration numbers shown in the registration number group 2 or an ortholog thereof in the test substance-added group is 2 times that of the test substance-free group.
If it is more than twice, it is evaluated that the test substance has anticancer activity.

Example 5 Preparation of Antitumor Activity Evaluation Cells Using Quantitative RT-PCR Cultured cells derived from ovarian cancer were seeded at 3.0 × 10 ⁵ cells per well of a 6-well plate (Sumitomo Bakelite Co., Ltd.). F
Culture using a BS-containing medium at 37 ° C. in the presence of 5% carbon dioxide for 2 nights. Next, the medium is exchanged with an FBS-containing medium to which a solution prepared by dissolving the test substance in 0.1% DMSO is added, and then the medium is cultured for 2 hours or 48 hours to obtain a cell culture solution of the test substance-added section. On the other hand, a cell culture solution containing no test substance is prepared by culturing in the same manner as above except that 0.1% DMSO is used instead of the test substance solution.

Gene Expression Analysis Total RNA was isolated from the cells of the test substance addition group and the cells of the non-addition group by the same procedure as in Example 1.
After the preparation, the cell-derived cDNAs of the test substance added group and the non-added group are prepared by the same operation as in Example 1. Next, using the prepared cDNA as a template, PCR is performed as described below to quantify the amplified DNA. That is, 1 μL of the cDNA, 20 pmol of the primer 3 having the nucleotide sequence shown by SEQ ID NO: 3, 20 pmol of the primer having the nucleotide sequence shown by SEQ ID NO: 4, and S
50 μL including 25 μL of YBR Green PCR Reagents PCR (ABI)
After preparing a reaction solution of, and using ABI7700 (ABI) for 2 minutes at 50 ° C and then 10 minutes at 95 ° C, 95 ° C for 15 seconds and then 60 ° C for 1 minute.
PCR is performed under the condition that 40 minutes of heat retention is performed for 1 cycle. From the amount of the amplified DNA, the expression level of this gene in the cells of the test substance-added group and the expression level of this gene in the cells of the test substance-free group are determined. When the expression level in the test substance-added group is 1/2 or less of the expression level in the test substance-free group, the test substance is evaluated to have anticancer activity.

Example 6 Search for Anti-Cancer Active Substance Using Quantitative RT-PCR In the same manner as in Example 5, a cell culture solution containing a test substance and a cell culture solution containing no test substance are prepared. . After preparing total RNA from the cells of the test compound-added group and the cells derived from the non-added group by the same operation as in Example 1, the test compound-added group and the non-added group were prepared by the same operation as in Example 1, respectively. CDNA from the cells of is prepared. Next, using the prepared cDNA as a template, PCR is performed as described below to quantify the amplified DNA. That is, 1 μL of the cDNA, 20 pmol of the primer 5 consisting of the nucleotide sequence represented by SEQ ID NO: 5, 20 pmol of the primer consisting of the nucleotide sequence represented by SEQ ID NO: 6, and S
50 μL including 25 μL of YBR Green PCR Reagents PCR (ABI)
After preparing a reaction solution of, and using ABI7700 (ABI) for 2 minutes at 50 ° C and then 10 minutes at 95 ° C, 95 ° C for 15 seconds and then 60 ° C for 1 minute.
PCR is performed under the condition that 40 minutes of heat retention is performed for 1 cycle. From the amount of the amplified DNA, the expression level of this gene in the cells of the test substance-added group and the expression level of this gene in the cells of the test substance-free group are determined. If the expression level in the test substance-added group is at least twice the expression level in the test substance-free group, it is evaluated that the test substance has anticancer activity.

[0050]

INDUSTRIAL APPLICABILITY The present invention can provide a method for detecting ovarian cancer cells, which uses an abnormal gene level as an index.

[Sequence Listing Free Text] SEQ ID NO: 1 Oligonucleotide primer designed for PCR SEQ ID NO: 2 Oligonucleotide primer designed for PCR SEQ ID NO: 3 Oligonucleotide primer designed for PCR SEQ ID NO: 4 Oligonucleotide Primer Designed for PCR SEQ ID NO: 5 Oligonucleotide Primer Designed for PCR SEQ ID NO: 6 Oligonucleotide Primer Designed for PCR

[0052]

[Sequence list] SEQUENCE LISTING <110> Sumitomo Chemical Co., Ltd. <120> Method for detecting Ovary Carcinoma Cells <130> P154225   <160> 6 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 1 ggagagttgc tgactttgta 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 2 tccaagtatg tctagtcacc 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 3 taggaggagc tgccacagtt 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 4 tttggctctg gcacaggctt 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 5 tccggacacc acaccacatt 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide primer for PCR <400> 6 ctgccatctc ttacttcagt 20

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 33/15 G01N 33/574 Z 33/50 C12N 15/00 ZNAA 33/574 A F term (reference) 2G045 AA26 AA29 AA40 BB20 CA25 CA26 CB01 CB03 CB17 DA12 DA13 DA14 DA78 FB02 FB05 FB07 4B024 AA01 AA12 CA04 CA09 CA12 CA20 HA11 HA13 HA14 4B063 QA01 QA05 QA19 QQ02 QQ21 QQ41 QQ53 QQ61 QQ89 QR08 QR32 QR35 QR40 QR42 QR55 QR62 QR77 QR84 QS25 QS28 QS34 QS39 QX02 4C084 AA17 NA14 ZB26

Claims (15)

[Claims] 1. A method for detecting ovarian cancer cells, which comprises (1)
A step of measuring an expression level in a sample of a gene having a nucleotide sequence registered in Genbank with any of the registration numbers shown in the following registration number groups 1 or 2 or one or more genes selected from the ortholog thereof,
(2) Comprising a step of comparing the measured expression level of the gene obtained in the step (1) with a control value of the expression level of the gene, and detecting ovarian cancer cells in the sample based on the difference A method characterized by the following. <Registration number group 1> X02344, AI884621, X59932, AI98943
2, X74929, AI346658, AI354351, AI332641, U28964, A
I672414, D26598, H44597, U65785, AA775776, AA74243
8, AI887311, M13560, AI807277, D38047, AI798743, X
04366, AI791182, H15313, AA004221, L24521, AI95497
5, D45248, AI973186, AF051782, AI762891, N37049, A
A554681, X57346, W21894, U48705, AA112121, AL05029
0, AW024537, W60640, AW024572, M26252, AA020846, U
85611, R54450, U78095, AI983181, AI741591, AI99112
2, W28869, AI147040, HG1614-HT1614, AI986430, AJ00
2308, AA062899, AA532392, AI560151, Y15915, AI3010
60, HG1980-HT2023, AI351790, M21186, AL041566, AI8
31655, AA211690, AI885170, AI241578, M91670, AW020
965, D42123, N32858, AI460118, AI539386, AI62038
1, AI804075, U21689, R20554, D89052, AI684451, AA4
79496, AA131307, X57348, AI992284, U77604, AI66041
2, AF054174, AI878836, AI991845, AW001374, AF00539
2, AI862775, U47634, AA928646, M83751, AI341234, A
I018296, AI394307, Z69043, AI032386, X00734, AI970
195, U49278, AI741313, AI634570, AI733988, AJ13118
2, AI613214, X67325, AW007442, AI653621, AA27853
8, T15777, AA543084, J03909, AA587236, L16842, AI5
57649, M34423, AI309320, AA622029, AI934540, Z9393
0, AA383718, X69699, AI979335, M65028, AA044793, A
I357616, AI417084, AF047436, AW009649, AW004750, A
I990317, AW007731, AI087923, AI867409, AI660570, A
F001601, N63815, AA004274, AI927408, K03515, AI624
028, AA576813, AI627731, M61832, AI923108, AW00950
5, AI969418, X73478, AI589365, AI458823, AW02600
3, Y13936, AI380624, AA913312, AI962313, AF04010
5, AI696971, AI264637, AI051599, AF029750, AI98416
6, AI971277, AI792635, X02152, AI286310, AI57183
0, AI928799, W28186, T15735, H98135, AI431799, M23
410, AA564822, AA156784, AA676723, M32578, AA12678
7, AW026119, AI635224, M55914, AI432513, AW00577
5, AI568057, S57501, AI817092, AI742737, AW01593
4, HG1112-HT1112, AI955339, AI986481, AW002819, AB
000095, AA857473, AI816843, AA908890, AI872092, AA
532655, AI743206, AI497609, AF016903, AA034414, AA
677666, AW002409 <Registration number group 2> M25079, AI669685, M69043, AL04829
7, AL050224, AW025584, H05010, AA602984, M12125, A
A497129, L27560, AI749098, Z11793, AI620911, AL046
979, AI983251, X59350, W51743, M96233, AI571452, U
58516, AI566871, AI814178, AI817897, L48215, AI688
582, T53591, AI357209, U77594, AA167094, AI97284
8, AI084193, U15979, AI656836, AI969659, R46698, A
B011170, AA115295, AI982873, R79768, AL050202, AL0
42569, N70077, AL042923, AF035292, N46649, AW01649
6, AA947305, AF037261, N21424, AW008412, AA17681
9, W28281, AI676103, AW003456, AI884831, U83411, H
16294, AW007987, AW001427, M62831, AI983913, H4161
4, AW022852, M99701, AI829724, AI916646, AL03736
3, S68805, AI684486, AI967936, H17681, AB003184, A
W009586, AI828593, AI610837, X77956, N92541, AW006
802, AA194540, L27560, AI146333, AI984087, AI35207
7, X86693, AL043598, AA281145, AI082237, D11428, A
I417267, AW007399, AI968892, M95787, AL043089, AA1
28061, AA143491, M33146, AW022607, AI290237, AI440
424, AL109701, AI769737, AI936699, AL037969, AL050
159, AI807023, R41648, AI983615, AL021154, AA78242
3, AW025487, N99196, X15880, AA194985, N24480, AW0
05790, J03507, AI985704, AA524029, W63545, AB00504
7, AW004042, AI810628, AW007869, AF013570, AW02273
5, AI262287, AI963008, AF052114, AA446219, AF06352
7, AW009564, L13720, R67627, AI075744, AA887130, A
F053944, R60248, AI553918, AW016563, U21128, AA227
065, AA625198, AI620412, M64497, W05842, AL04284
2, AA838843, X52022, W70067, N45224, AW015393, M14
058, AI934584, AI279875, AI922443, AI557295, AA528
255, AA131626, AI208816, X54486, AA503073, AA15016
5, AW007532, X79683, AI031686, AI066756, AW00648
5, U20982, AI858626, AI752406, AA101758, J02854, A
I698134, AI338338, AI004969, U18009, AA906378, AA5
95199, AI979262, U69559, AI125667, AW023627, H4208
5, AL021366, AA146966, AL039400, AI985751, U2239
8, AA521416, AI123567, N50065, M92843, AI016714, A
I564314, AI073544, D78134, AA643513, AI340029, AI8
21126, J04080, AA524436, AL046946, AI300876, AF032
885, AI338536, W73819, AI660901, AB017019, N7813
9, W30943, AI792487, U97105, AI246018, W27481, R54
619, AF049910, AI142548, AI913548, AI333006, AL035
494, AI970823, AI670862, AA897407, U29953, AW05152
7, AA430300, AI376179, W26496, AA523172, AA41138
2, AI418353, AB023209, AI635057, AI928515, AI91743
2, AI651806, AI830095, AI971235, AI022831, Z2255
5, N68231, AA102468, AI092890, X72889, AA778836, A
I860150, AI381576, AA149307, AI982754, AA946755, A
I421663, AI743134, AI887362, W55861, AI557412, AL0
50223, AA479898, AL047415, AA204642, AL046940, AW0
25350, AI688114, AI762319, X13839, AA460952, AA044
844, AI799496, AF055376, AI355848, AI300073, AA147
088, X51345, AI983649, AA526844, AI972597, Y1362
2, AA587245, AI860250, AI890480, AB007898, AW00894
1, AW016298, AI651760, M29039, N25612, AA843523, A
I525812, AL049381, AA557557, N24239, AW021895, V01
512, N66434, AI359214, H67928, D15050, AI990337, A
I810627, AI825981, M62403, AA628405, AI796967, AF0
22813, AI872510, AI052110, AI734967, U17714, AA214
086, AI535730, W52024, AI979070, W63773, AA62147
5, L13720, AI810399, AA531016 2. The method according to claim 1, wherein the expression level of the gene is measured by measuring the transcript amount of the gene. 3. The method according to claim 1 or 2, wherein the control value of the expression level of the gene is the value of the expression level of the gene in normal tissues or normal cells. 4. In the step (2), (a) registration number group 1
It is shown in (b) Accession number group 2 using as an index the measured value of the expression level of the gene or its ortholog having the nucleotide sequence registered in Genbank with any of the accession numbers shown in (b) as an index. The ovarian cancer cell in a sample is detected using as an index that the measured value of the expression level of the gene having a nucleotide sequence registered in Genbank with any of the registration numbers or its ortholog is lower than the control value. The method described. 5. A method for evaluating the anticancer activity of a substance, which comprises:
(1) a step of bringing an ovarian cancer tissue or ovarian cancer cell into contact with a test substance, (2) the above-mentioned registration number group 1 or 2 in the tissue or cell brought into contact with the test substance in the step (1) Gen with any of the registration numbers shown
a step of measuring the expression level of one or more genes selected from genes having a nucleotide sequence registered in bank or an ortholog thereof, and (3) the measured value of the expression level of the gene obtained in the step (2) A method comprising a step of comparing the expression level of the gene with a control value and evaluating the anticancer activity of the test substance based on the difference. 6. The method according to claim 5, wherein the expression level of the gene is measured by measuring the transcript amount of the gene. 7. The method according to claim 5, wherein the control value of the expression level of the gene is the value of the expression level of the gene in ovarian cancer tissue or ovarian cancer cells. 8. In the step (3), (a) registration number group 1
An index is that the measured value of a gene having a nucleotide sequence registered in Genbank with any of the accession numbers shown in 1 or its ortholog is lower than the control value, (b)
Genb with any of the registration numbers shown in registration number group 2
The method according to claim 7, wherein the anticancer activity of the test substance is evaluated by using, as an index, the measured value of the expression level of the gene having the nucleotide sequence registered in ank or its ortholog is higher than the control value. 9. An anticancer active substance characterized by comprising a step of selecting a substance having an anticancer activity based on the anticancer activity evaluated by the method according to any one of claims 5 to 8. Search method. 10. An anticancer agent containing an anticancer active substance selected by the search method according to claim 9 as an active ingredient. 11. A method for evaluating carcinogenic activity of a substance, comprising:
(1) a step of contacting a tissue or cell derived from ovary with a test substance, (2) registration of the tissue or cell contacted with the test substance in the step (1) shown in the above registration number groups 1 or 2 Genban by any of the numbers
the step of measuring the expression level of one or more genes selected from the genes having the nucleotide sequence registered in k or its ortholog, and (3) the measured value of the expression level of the gene obtained in step (2) above. A method comprising the step of comparing the expression level of the gene with a control value and evaluating the carcinogenic activity of the test substance based on the difference. 12. The method according to claim 11, wherein the expression level of the gene is measured by measuring the transcript amount of the gene. 13. The method according to claim 11 or 12, wherein the control value of the expression level of the gene is the value of the expression level of the gene in normal tissues or normal cells. 14. In step (3), (a) the measured value of a gene having a nucleotide sequence registered in Genbank with any of the accession numbers shown in accession number group 1 or its ortholog is higher than the control value. With that as an index,
(B) G in any of the registration numbers shown in the registration number group 2
The method according to claim 13, wherein the carcinogenic activity of the test substance is evaluated by using, as an index, the measured value of the expression level of the gene having the nucleotide sequence registered in enbank or its ortholog is lower than the control value. 15. A method for searching a carcinogenic substance, comprising the step of selecting a substance having a carcinogenic activity based on the carcinogenic activity evaluated by the method according to claim 11.