JP3713461B2 - Estrogen activity inhibitory effect evaluation method using gene expression as an index - Google Patents

Description

【００９４】
表1の説明
タモキシフェン効果およびタモキシフェンによるエストロゲン活性抑制効果の評価に供した遺伝子またはESTの全リスト。27の対照遺伝子またはESTは陰影で示した。
【００９５】
（各カラムの説明）
Gene Name：データベース（GenBank, EMBL, DDBJ）に登録されている名前。ただし、省略形、通称名も含む。
Accession No.：上記データベースにおけるアクセッション番号。
10nM E2 Raw DATA：MCF7細胞に10nMエストロゲンを作用させた時の値。
10nM E2 +5μM TAM Raw DATA：MCF7細胞に10nMエストロゲンと5μMタモキシフェン（TAM）を同時に作用させた時の値。
10nM E2 / 10nM E2 +5μM TAM：MCF7細胞に10nMエストロゲンを作用させた時の値とMCF7細胞に10nMエストロゲンと5μMタモキシフェン（TAM）を同時に作用させた時の値の商。
10nM TAM：10nMタモキシフェン（TAM）のみをMCF7細胞に作用させた時の値。
【００９６】
【表２】

【００９７】
表2の説明
表1を以下の基準に従って各遺伝子またはESTを19項目に分類した。１）表1において（10nM E2 / 10nM E2 +5μM TAM）の値が3.00以上のもの、すなわちエストロゲン10nMとタモキシフェン5μMを競合させてMCF-7細胞に作用させた結果から、タモキシフェン5μMによって強く抑制されたもの、（10nM E2 / 10nM E2 +5μM TAM）の値が2.99以下1.50以上のもの、すなわちやや抑制されたもの、（10nM E2 / 10nM E2 +5μM TAM）の値が1.49以下、-1.49以上のもの、すなわち変化の無かったもの、（10nM E2 / 10nM E2 +5μM TAM）の値が-1.50以下 -1.90以上のもの、すなわちやや誘導されたもの、（10nM E2 / 10nM E2 +5μM TAM）の値が-2.00以下のもの、すなわち強く誘導されたもの。
【００９８】
１）上で分類した項目それぞれをタモキシフェン10nMだけをMCF-7細胞に作用させた結果から次の基準に従って更に分類した。表1において（10nM TAM）の値が-2.00以下のもの、すなわちタモキシフェン10nMによって強く抑制されたもの、（10nM TAM）の値が-1.90以上-1.50以下のもの、すなわちタモキシフェン10nMによってやや抑制されたもの、（10nM TAM）の値が-1.49以上1.49以下のもの、すなわちタモキシフェン10nMによって変化のないもの、（10nM TAM）の値が1.50以上1.99以下のもの、すなわちタモキシフェン10nMによってやや誘導されたもの、（10nM TAM）の値が2.00以上のもの、すなわちタモキシフェン10nMによって強く誘導されたもの。この基準に従って分類した結果合計19項目に分類された。
【００９９】
【発明の効果】
実施例に示したように、本発明の遺伝子またはESTはエストロゲンまたはタモキシフェンで刺激した場合に特定の発現パターンを示す。被験物質によりこれらの遺伝子またはESTの発現が、エストロゲンの存在下または非存在下に抑制されるかまたは誘導されるかを調べることにより、被験物質がエストロゲン活性を抑制するか、タモキシフェン類似活性を有するか否かを決定することができる。
【０１００】
【配列表】

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for evaluating the effect of tamoxifen on the estrogen activity, a method for evaluating the effect of tamoxifen, using DNA containing all or part of the base sequence of an estrogen-responsive gene or EST (Expressed sequence tag) And a method for evaluating whether an unknown test substance has a tamoxifen-like action.
[0002]
[Prior art]
Estrogens are steroidal female hormones that are known to exert diverse physiological activities on many organs such as the mammary gland and uterus, and to promote carcinogenesis and cancer progression in some organs. .
On the other hand, tamoxifen is a triphenylethylene derivative, which is known to bind to the estrogen receptor and have estrogen antagonist activity. Tamoxifen exhibits an anti-estrogen action against estrogen receptors such as breast cancer and exerts an anti-breast cancer action.
However, the mechanism of action of estrogens has not been fully elucidated because the mechanism of action of estrogens is very complex through multiple pathways. In addition, the effects of tamoxifen have not been fully elucidated.
[0003]
Elucidation of the mechanism of action of estrogen and tamoxifen is desired for the development of anticancer drugs, analysis of environmental hormones, etc. It was desired to establish a method for evaluation. In particular, the estrogen activity inhibitory effect is an important criterion for screening anti-cancer drugs, etc., but there are evaluation methods based on fluctuations in the expression of a single gene or a very small number of genes. There was still no evaluation method used. Therefore, there has been a need for an evaluation method that enables the development and screening of drugs that exhibit the same structure or action as tamoxifen and that have higher action and lower side effects.
[0004]
[Problems to be solved by the invention]
The present invention relates to a method for evaluating the estrogen activity inhibitory effect of tamoxifen, a method for evaluating the effect of tamoxifen, an unknown test substance, using DNA containing all or part of an estrogen-responsive gene or EST base sequence It is an object of the present invention to provide a method for evaluating whether or not has a tamoxifen-like action, and a DNA microarray containing DNA containing all or part of the base sequence of the gene or EST for the evaluation.
[0005]
[Means for Solving the Problems]
Tamoxifen has anticancer activity as an antagonist of estrogen, but when evaluating its effect using gene expression, not only is there a gene response due to the effect of suppressing estrogen activity, but it also originates from reactivity such as structure and metabolism It is difficult to make accurate judgments because it includes gene responses specific to tamoxifen. Therefore, in order to evaluate the effect of tamoxifen using a gene group related to the estrogen activity suppression effect, it is necessary to know the effect on the gene unique to tamoxifen. In particular, in order to clarify the gene group involved in the mechanism of estrogen action, regarding the estrogen activity inhibitory effect and tamoxifen's unique effect, we clarified common genes, common genes with different degrees of effect, and non-common genes, It is necessary to create an evaluation method (screening method) using it.
[0006]
Based on these ideas, the present inventors searched for genes or ESTs whose expression varies when stimulated with estrogen, and further, using a cDNA microarray, when estrogen and / or tamoxifen are stimulated simultaneously. Variations in the expression of the gene or EST were examined. As a result, we found that there are many genes whose expression levels fluctuate, and these genes have different expression patterns depending on the stimulation amount of estrogen and tamoxifen. The present inventors have found that there are genes whose expression is suppressed when cells are stimulated with estrogen and / or tamoxifen, and genes whose expression is induced. I found. Furthermore, unknown substances that exhibit estrogen activity inhibitory effect by the same mechanism as that of tamoxifen are expected to show the same gene response as tamoxifen. It is found that the same effect as the substance tamoxifen can be evaluated, a method for evaluating the effect of tamoxifen on estrogen activity using these genes, a method for evaluating the effect of tamoxifen, and an unknown test substance using these genes The present inventors have completed the present invention relating to a method for evaluating the effects of tamoxifen, that is, a method for screening a substance having a tamoxifen-like action.
[0007]
That is, the present invention is as follows.
(1) Among the genes or ESTs whose expression is affected by a chemical substance having an estrogen-like activity, the expression is suppressed compared to the case where only a chemical substance having an estrogen-like activity exists when estrogen and tamoxifen are mixed. A method for evaluating the inhibitory effect of tamoxifen on estrogen activity using a DNA comprising all or part of a base sequence of a gene or EST that is induced or induced.
[0008]
(2) Among the genes or ESTs whose expression is affected by a chemical substance having estrogen-like activity, the expression is suppressed compared to the case where only a chemical substance having estrogen-like activity is present when estrogen and tamoxifen are mixed. Or analyzing the expression of the gene using a DNA microarray spotted with a DNA containing all or part of the base sequence of the gene or EST to be induced or induced.
[0009]
(3) Among the genes or ESTs whose expression is affected by a chemical substance having an estrogen-like activity, the expression is suppressed compared to the case where only a chemical substance having an estrogen-like activity is present when estrogen and tamoxifen are mixed. Or analyzing the expression of the gene by real-time PCR targeting the gene or EST that is induced or induced.
[0010]
(4) Among the genes or ESTs whose expression is affected by a chemical substance having an estrogen-like activity, the expression is suppressed compared to the case where only a chemical substance having an estrogen-like activity is present when estrogen and tamoxifen are mixed. A group consisting of A1 to A15 containing the following class 1, class 2 and class 3 genes or ESTs and B consisting of B1 to B7 containing the classes 17, 18 and 19 genes or ESTs. The method according to any one of (1) to (3), wherein DNA comprising all or a part of one or more genes of the group or the base sequence of EST is used.
[0011]
(Category 1) Strongly suppressed by 5 μM tamoxifen due to competition with estrogen, but slightly induced only by 10 nM tamoxifen
(A1) Homo sapiens methylene tetrahydrofolate dehydrogenase (XM-002234) (SEQ ID NO: 1),
(Category 2) Strongly suppressed by 5μM tamoxifen due to competition with estrogen, but no change with 10nM tamoxifen alone
(A2) asparagine synthetase (GenBank accession number L35946) (SEQ ID NO: 2)
(A3) EST (GenBank accession number AA587912) (SEQ ID NO: 3),
(A4) exportin (GenBank accession number AF039022) (SEQ ID NO: 4),
(A5) ferritin, heavy polypeptide 1 (GenBank accession number AA102267) (SEQ ID NO: 5),
(A6) glutamine-fructose-6-phosphate transaminase 1 (GenBank accession number M90516) (SEQ ID NO: 6),
(A7) 78KD GLUCOSE REGULATED PROTEIN PRECURSOR (GenBank accession number AI878886) (SEQ ID NO: 7),
(A8) Homo sapiens partial mRNA for A-type potassium channel modulatory protein 2 (GenBank accession number AJ276317) (SEQ ID NO: 8),
(A9) insulin-like growth factor-binding protein 4 (GenBank accession number M62403) (SEQ ID NO: 9),
(A10) methylene tetrahydrofolate dehydrogenase (GenBank accession number X16396) (SEQ ID NO: 10),
(A11) phosphoenolpyruvate carboxykinase 2 (GenBank accession number X92720) (SEQ ID NO: 11),
(A12) protein kinase C, delta (GenBank accession number D10495) (SEQ ID NO: 12),
(A13) trefoil factor 1 (GenBank accession number X05030) (SEQ ID NO: 13),
(Category 3) Strongly suppressed by 5 μM tamoxifen due to competition with estrogen, but slightly suppressed only by 10 nM tamoxifen
(A14) argininosuccinate synthetase (GenBank accession number AI660571)
(SEQ ID NO: 14),
(A15) solute carrier family 7, member 5 (GenBank accession number XM-017410) (SEQ ID NO: 15),
(Category 17) Strongly induced by 5 μM tamoxifen in competition with estrogen, with no change with 10 nM tamoxifen alone
(B1) erbB2 (GenBank accession number M86910) (SEQ ID NO: 16),
(B2) fucosyltransferase 8 (GenBank accession number Y17977) (SEQ ID NO: 17),
(B3) Homo sapiens, adipose specific 2 (GenBank accession number BC004471) (SEQ ID NO: 18),
(B4) insulin-like growth factor binding protein 5 (GenBank accession number L27556) (SEQ ID NO: 19),
(Category 18) Strongly induced by 5 μM tamoxifen due to competition with estrogen and slightly suppressed by 10 nM tamoxifen alone
(B5) Homo sapiens DNA from chromosome 19 (GenBank accession number AC004030) (SEQ ID NO: 20),
(B6) quiescin Q6 (GenBank accession number U97276) (SEQ ID NO: 21), and
(Category 19) Strongly induced by 5 μM tamoxifen in competition with estrogen and strongly suppressed by 10 nM tamoxifen alone
(B7) solute carrier family 12, member 2 (GenBank accession number XM-003745) (SEQ ID NO: 22)
[0012]
(5) A method for evaluating the effect of tamoxifen using a DNA containing all or part of the base sequence of a gene or EST whose expression is affected by a chemical substance having tamoxifen-like activity.
(6) The analysis of the expression of the gene using a DNA microarray spotted with a gene having a tamoxifen-like activity, or a DNA that includes all or part of the EST base sequence, the expression of which is affected. The method of (5).
[0013]
(7) The method according to (5), comprising analyzing the expression of a gene whose expression is affected by a chemical substance having tamoxifen-like activity or real-time PCR targeting EST.
(8) The C group consisting of C1 to C11 including the following class 12 and class 16 genes or ESTs whose expression is affected by a chemical substance having tamoxifen-like activity, and including the group 8 and class 13 genes or ESTs The method according to any one of (5) to (7), wherein DNA comprising all or part of one or more genes of D group consisting of D1 to D8 or the base sequence of EST is used.
[0014]
(Category 12) Competing with estrogen, no change with 5 μM tamoxifen, strong suppression with 10 nM tamoxifen alone
(C1) A1B1 (GenBank accession number AF137620) (SEQ ID NO: 23),
(C2) Estrogen receptor α (GenBank accession number AY033393) (SEQ ID NO: 24),
(C3) Homo sapiens UDP-Gal: betaGlcNAc beta 1,4-galactocyltransferase, polypeptide1 (GenBank accession number NM-001497) (SEQ ID NO: 25),
(C4) stearoyl-CoA desaturase (GenBank accession number XM-005719) (SEQ ID NO: 26),
(Category 16) Induced somewhat by 5μM tamoxifen due to competition with estrogen and strongly suppressed only by 10nM tamoxifen
(C5) cadherin 18 (GenBank accession number NM-004934) (SEQ ID NO: 27),
(C6) enolase 2 (GenBank accession number X51956) (SEQ ID NO: 28),
(C7) enolase 3 (GenBank accession number X56832) (SEQ ID NO: 29),
(C8) Human mRNA for KIAA0373 gene (GenBank accession number AB002371) (SEQ ID NO: 30),
(C9) Homo sapiens tumor-associated calcium signal transducer 2 (GenBank accession number XM-001710) (SEQ ID NO: 31),
(C10) Homo sapiens enolase 1 (alpha) (ENO1) (GenBank accession number NM-001428) (SEQ ID NO: 32), and
(C11) selenium binding protein 1 (GenBank accession number U29091) (SEQ ID NO: 33),
(Category 8) Competing with estrogen, no change with 5 μM tamoxifen, strongly induced with 10 nM tamoxifen alone
(D1) CDC6 homolog (GenBank accession number NM-001254) (SEQ ID NO: 34),
(D2) chromobox homolog 1 (GenBank accession number BC002609) (SEQ ID NO: 35),
(D3) Homo sapiens interleukin 2 receptor, beta (GenBank accession number XM-009962) (SEQ ID NO: 36),
(D4) Human mRNA for KIAA0008 (GenBank accession number D13633) (SEQ ID NO: 37),
(D5) Human mRNA for KIAA0101 gene (GenBank accession number D14657) (SEQ ID NO: 38),
(D6) KIAA1051 protein (GenBank Accession No. AB028974) (SEQ ID NO: 39)
(Category 13) Slightly induced by 5 μM tamoxifen due to competition with estrogen, but strongly induced only by 10 nM tamoxifen
(D7) Homo sapiens paired basic amino acid cleaving system 4 (GenBank accession number NM-002570) (SEQ ID NO: 40), and
(D8) neuropeptide Y receptor Y1 (GenBank accession number NM-000909) (SEQ ID NO: 41)
[0015]
(9) Tamoxifen using DNA comprising all or part of one or more genes of A1 to A15 and B1 to B7 of (4) and C1 to C11 and D1 to D8 of (8) or the base sequence of EST To evaluate the effect of the.
(10) DNA for evaluating the estrogen activity inhibitory effect of tamoxifen, spotted with DNA comprising one or more genes of A1 to A15 and B1 to B7 of (4) or a portion of the EST base sequence. Microarray.
[0016]
(11) A DNA microarray for evaluating the effect of tamoxifen on which one or more genes or ESTs of C1 to C11 and D1 to D8 of (8) are spotted.
(12) DNA comprising one or more genes of A1 to A15 and B1 to B7 of (4) and one or more genes of C1 to C11 and D1 to D8 of (8) or a base sequence of EST was spotted. DNA microarray to evaluate the effects of tamoxifen.
[0017]
(13) When estrogen and tamoxifen coexist, a gene whose expression is suppressed as compared with the case where only a chemical substance having an estrogen-like activity is present or DNA containing all or part of the base sequence of EST is used. Alternatively, a method of screening for a substance having an action of suppressing EST response to estrogen in the same manner as tamoxifen.
[0018]
(14) From the A1 to A15 containing genes or ESTs of the following classification 1, classification 2 and classification 3 whose expression is suppressed when estrogen and tamoxifen coexist compared to the case where only a chemical substance having an estrogen-like activity is present The method according to (13), wherein DNA comprising all or a part of one or more genes of the group A or EST base sequence is used.
[0019]
(Category 1) Strongly suppressed by 5 μM tamoxifen due to competition with estrogen, but slightly induced only by 10 nM tamoxifen
(A1) Homo sapiens methylene tetrahydrofolate dehydrogenase (XM-002234) (SEQ ID NO: 1),
(Category 2) Strongly suppressed by 5μM tamoxifen due to competition with estrogen, but no change with 10nM tamoxifen alone
(A2) asparagine synthetase (GenBank accession number L35946) (SEQ ID NO: 2)
(A3) EST (GenBank accession number AA587912) (SEQ ID NO: 3),
(A4) exportin (GenBank accession number AF039022) (SEQ ID NO: 4),
(A5) ferritin, heavy polypeptide 1 (GenBank accession number AA102267) (SEQ ID NO: 5),
(A6) glutamine-fructose-6-phosphate transaminase 1 (GenBank accession number M90516) (SEQ ID NO: 6),
(A7) 78KD GLUCOSE REGULATED PROTEIN PRECURSOR (GenBank accession number AI878886) (SEQ ID NO: 7),
(A8) Homo sapiens partial mRNA for A-type potassium channel modulatory protein 2 (GenBank accession number AJ276317) (SEQ ID NO: 8),
(A9) insulin-like growth factor-binding protein 4 (GenBank accession number M62403) (SEQ ID NO: 9),
(A10) methylene tetrahydrofolate dehydrogenase (GenBank accession number X16396) (SEQ ID NO: 10),
(A11) phosphoenolpyruvate carboxykinase 2 (GenBank accession number X92720) (SEQ ID NO: 11),
(A12) protein kinase C, delta (GenBank accession number D10495) (SEQ ID NO: 12),
(A13) trefoil factor 1 (GenBank accession number X05030) (SEQ ID NO: 13),
(Category 3) Strongly suppressed by 5 μM tamoxifen due to competition with estrogen, but slightly suppressed only by 10 nM tamoxifen
(A14) argininosuccinate synthetase (GenBank accession number AI660571) (SEQ ID NO: 14),
(A15) solute carrier family 7, member 5 (GenBank accession number XM-017410) (SEQ ID NO: 15)
[0020]
(15) Analyzing the expression of the gene using a DNA microarray spotted with a DNA comprising one or more genes of A group consisting of A1 to A15 or EST of (14) or DNA containing all or part of the base sequence of EST (14) The method of including.
(16) The method according to (14), comprising analyzing expression of the gene by real-time PCR targeting one or more genes of group A consisting of the genes A1 to A15 or EST of (14) or EST.
[0021]
(17) The gene or EST responding to estrogen spotted by DNA comprising all or part of one or more genes of group A consisting of the genes A1 to A15 or EST of (14) or the EST base sequence. A DNA microarray for screening substances that have the same inhibitory action as tamoxifen.
[0022]
(18) When estrogen and tamoxifen coexist, a gene whose expression is induced compared to the case where only a chemical substance having an estrogen-like activity is present or a DNA containing all or part of the base sequence of EST, Alternatively, a method for screening a substance having an action of inducing EST to respond to estrogen in the same manner as tamoxifen.
[0023]
(19) When the estrogen and tamoxifen coexist, the expression is induced as compared to the case where only a chemical substance having an estrogen-like activity is present. (18) The method according to (18), wherein one or more genes of group B consisting of a gene and EST or DNA containing all or part of the base sequence of EST is used.
[0024]
(Category 17) Strongly induced by 5 μM tamoxifen in competition with estrogen, with no change with 10 nM tamoxifen alone
(B1) erbB2 (GenBank accession number M86910) (SEQ ID NO: 16),
(B2) fucosyltransferase 8 (GenBank accession number Y17977) (SEQ ID NO: 17),
(B3) Homo sapiens, adipose specific 2 (GenBank accession number BC004471) (SEQ ID NO: 18),
(B4) insulin-like growth factor binding protein 5 (GenBank accession number L27556) (SEQ ID NO: 19),
(Category 18) Strongly induced by 5 μM tamoxifen due to competition with estrogen and slightly suppressed by 10 nM tamoxifen alone
(B5) Homo sapiens DNA from chromosome 19 (GenBank accession number AC004030) (SEQ ID NO: 20),
(B6) quiescin Q6 (GenBank accession number U97276) (SEQ ID NO: 21), and
(Category 19) Strongly induced by 5 μM tamoxifen in competition with estrogen and strongly suppressed by 10 nM tamoxifen alone
(B7) solute carrier family 12, member 2 (GenBank accession number XM-003745) (SEQ ID NO: 22)
[0025]
(20) Expression of the gene using a DNA microarray spotted with DNA containing all or part of one or more genes of group B consisting of B1-B7 genes or ESTs of (19) or EST base sequences The method according to (19), comprising analyzing.
(21) The method according to (19), which comprises analyzing expression of the gene by real-time PCR targeting one or more genes of group B consisting of the B1-B7 gene or EST of (19) or EST.
[0026]
(22) The gene or EST responding to estrogen by spotting DNA containing all or part of one or more genes of the B group consisting of the B1-B7 gene or EST of (19) or the EST base sequence. A DNA microarray for screening substances having an effect of inducing the same as tamoxifen.
[0027]
(23) When estrogen and tamoxifen are mixed, a gene whose expression is induced or suppressed compared to the case where only a chemical substance having an estrogen-like activity is present or DNA containing all or part of the base sequence of EST, A method of screening for a substance having an action of inducing or inhibiting the gene or EST from responding to estrogen in the same manner as tamoxifen.
[0028]
(24) In the case where estrogen and tamoxifen coexist, the expression is induced or suppressed as compared with the case where only a chemical substance having an estrogen-like activity is present. A group consisting of A15 and a DNA comprising one or more genes of group B consisting of B1 to B7 including genes or ESTs of classes 17, 18 and 19 or EST, or DNA containing all or part of the base sequence of EST (23 )the method of.
[0029]
(Category 1) Strongly suppressed by 5 μM tamoxifen due to competition with estrogen, but slightly induced only by 10 nM tamoxifen
(A1) Homo sapiens methylene tetrahydrofolate dehydrogenase (XM_002234) (SEQ ID NO: 1),
(Category 2) Strongly suppressed by 5μM tamoxifen due to competition with estrogen, but no change with 10nM tamoxifen alone
(A2) asparagine synthetase (GenBank accession number L35946) (SEQ ID NO: 2)
(A3) EST (GenBank accession number AA587912) (SEQ ID NO: 3),
(A4) exportin (GenBank accession number AF039022) (SEQ ID NO: 4),
(A5) ferritin, heavy polypeptide 1 (GenBank accession number AA102267) (SEQ ID NO: 5),
(A6) glutamine-fructose-6-phosphate transaminase 1 (GenBank accession number M90516) (SEQ ID NO: 6),
(A7) 78KD GLUCOSE REGULATED PROTEIN PRECURSOR (GenBank accession number AI878886) (SEQ ID NO: 7),
(A8) Homo sapiens partial mRNA for A-type potassium channel modulatory protein 2 (GenBank accession number AJ276317) (SEQ ID NO: 8),
(A9) insulin-like growth factor-binding protein 4 (GenBank accession number M62403) (SEQ ID NO: 9),
(A10) methylene tetrahydrofolate dehydrogenase (GenBank accession number X16396) (SEQ ID NO: 10),
(A11) phosphoenolpyruvate carboxykinase 2 (GenBank accession number X92720) (SEQ ID NO: 11),
(A12) protein kinase C, delta (GenBank accession number D10495) (SEQ ID NO: 12),
(A13) trefoil factor 1 (GenBank accession number X05030) (SEQ ID NO: 13),
(Category 3) Strongly suppressed by 5 μM tamoxifen due to competition with estrogen, but slightly suppressed only by 10 nM tamoxifen
(A14) argininosuccinate synthetase (GenBank accession number AI660571) (SEQ ID NO: 14),
(A15) solute carrier family 7, member 5 (GenBank accession number XM-017410) (SEQ ID NO: 15),
(Category 17) Strongly induced by 5 μM tamoxifen in competition with estrogen, with no change with 10 nM tamoxifen alone
(B1) erbB2 (GenBank accession number M86910) (SEQ ID NO: 16),
(B2) fucosyltransferase 8 (GenBank accession number Y17977) (SEQ ID NO: 17),
(B3) Homo sapiens, adipose specific 2 (GenBank accession number BC004471) (SEQ ID NO: 18),
(B4) insulin-like growth factor binding protein 5 (GenBank accession number L27556) (SEQ ID NO: 19),
(Category 18) Strongly induced by 5 μM tamoxifen due to competition with estrogen and slightly suppressed by 10 nM tamoxifen alone
(B5) Homo sapiens DNA from chromosome 19 (GenBank accession number AC004030) (SEQ ID NO: 20),
(B6) quiescin Q6 (GenBank accession number U97276) (SEQ ID NO: 21), and
(Category 19) Strongly induced by 5 μM tamoxifen in competition with estrogen and strongly suppressed by 10 nM tamoxifen alone
(B7) solute carrier family 12, member 2 (GenBank accession number XM-003745) (SEQ ID NO: 22)
[0030]
(25) A DNA comprising all or part of one or more genes of group A consisting of A1 to A15 or EST of (24) and one or more genes of group B consisting of B1 to B7 genes or EST, or EST base sequence The method according to (24), comprising analyzing expression of the gene using a spotted DNA microarray.
(26) Expression of the gene by real-time PCR targeting one or more genes of group A consisting of A1 to A15 or EST of (24) and one or more genes of group B consisting of B1 to B7 genes or EST or EST The method according to (24), comprising analyzing.
[0031]
(27) A DNA comprising all or part of one or more genes of group A consisting of A1 to A15 or EST of (24) and one or more genes of group B consisting of B1 to B7 genes or EST, or the base sequence of EST A DNA microarray for screening substances that have the effect of inducing or suppressing the spotted gene or EST response to estrogen in the same manner as tamoxifen.
(28) Screening for a substance that suppresses the expression of the gene or EST in the same manner as tamoxifen, using a DNA that contains all or part of the base sequence of the gene or EST whose expression is suppressed by a chemical substance having tamoxifen-like activity Method.
[0032]
(29) All of the nucleotide sequences of one or more genes or ESTs of group C consisting of C1 to C11 genes including the following class 12 and class 16 genes or ESTs whose expression is suppressed by a chemical substance having tamoxifen-like activity Or the method of (28) using DNA containing a part.
[0033]
(Category 12) Competing with estrogen, no change with 5 μM tamoxifen, strong suppression with 10 nM tamoxifen alone
(C1) A1B1 (GenBank accession number AF137620) (SEQ ID NO: 23),
(C2) Estrogen receptor α (GenBank accession number AY033393) (SEQ ID NO: 24),
(C3) Homo sapiens UDP-Gal: betaGlcNAc beta 1,4-galactocyltransferase, polypeptide1 (GenBank accession number NM-001497) (SEQ ID NO: 25),
(C4) stearoyl-CoA desaturase (GenBank accession number XM-005719) (SEQ ID NO: 26),
(Category 16) Induced somewhat by 5μM tamoxifen due to competition with estrogen and strongly suppressed only by 10nM tamoxifen
(C5) cadherin 18 (GenBank accession number NM-004934) (SEQ ID NO: 27),
(C6) enolase 2 (GenBank accession number X51956) (SEQ ID NO: 28),
(C7) enolase 3 (GenBank accession number X56832) (SEQ ID NO: 29),
(C8) Human mRNA for KIAA0373 gene (GenBank accession number AB002371) (SEQ ID NO: 30),
(C9) Homo sapiens tumor-associated calcium signal transducer 2 (GenBank accession number XM-001710) (SEQ ID NO: 31),
(C10) Homo sapiens enolase 1 (alpha) (ENO1) (GenBank accession number NM-001428) (SEQ ID NO: 32), and
(C11) selenium binding protein 1 (GenBank accession number U29091) (SEQ ID NO: 33)
[0034]
(30) Analysis of the expression of the gene using a DNA microarray spotted with one or more genes of C group consisting of C1 to C11 of (29) or DNA containing all or part of the base sequence of EST. (29) The method of including.
(31) The method according to (29), comprising analyzing expression of the gene by real-time PCR targeting one or more genes of group C consisting of the C1-C11 gene or EST of (29) or EST.
(32) Expression of the gene or EST spotted with tamoxifen by spotting DNA containing all or part of one or more genes of the C group consisting of the C1-C11 gene or EST of (29) or the EST base sequence Similarly, DNA microarray for screening substances to be suppressed.
[0035]
(33) Screening for a substance that induces expression of the gene or EST in the same manner as tamoxifen, using a gene containing the whole or part of the base sequence of the gene or EST whose expression is induced by a chemical substance having tamoxifen-like activity Method. (34) all or one or more base sequences of one or more genes or ESTs of group D consisting of D1 to D8 including the following class 8 and class 13 genes or ESTs whose expression is induced by a chemical substance having tamoxifen-like activity The method according to (33), wherein a DNA containing a part thereof is used.
[0036]
(Category 8) Competing with estrogen, no change with 5 μM tamoxifen, strongly induced with 10 nM tamoxifen alone
(D1) CDC6 homolog (GenBank accession number NM-001254) (SEQ ID NO: 34),
(D2) chromobox homolog 1 (GenBank accession number BC002609) (SEQ ID NO: 35),
(D3) Homo sapiens interleukin 2 receptor, beta (GenBank accession number XM-009962) (SEQ ID NO: 36),
(D4) Human mRNA for KIAA0008 (GenBank accession number D13633) (SEQ ID NO: 37),
(D5) Human mRNA for KIAA0101 gene (GenBank accession number D14657) (SEQ ID NO: 38),
(D6) KIAA1051 protein (GenBank Accession No. AB028974) (SEQ ID NO: 39)
(Category 13) Slightly induced by 5 μM tamoxifen due to competition with estrogen, but strongly induced only by 10 nM tamoxifen
(D7) Homo sapiens paired basic amino acid cleaving system 4 (GenBank accession number NM-002570) (SEQ ID NO: 40), and
(D8) neuropeptide Y receptor Y1 (GenBank accession number NM-000909) (SEQ ID NO: 41)
[0037]
(35) Analyzing the expression of the gene using a DNA microarray spotted with DNA comprising all or part of one or more genes of the D group consisting of the D1-D8 genes or ESTs of (34) or EST base sequences. (34) The method of including.
(36) The method according to (34), comprising analyzing expression of the gene by real-time PCR targeting one or more genes or ESTs of the D group consisting of the genes D1 to D8 or EST of (34).
[0038]
(37) The expression of the gene or EST obtained by spotting DNA containing all or part of one or more genes of the D group consisting of the genes D1 to D8 or the EST of (34) or the base sequence of the EST, and tamoxifen Similarly, DNA microarray for screening substances to induce.
(38) A substance that acts in the same manner as tamoxifen on the expression of a gene or EST using a gene or the DNA whose expression is suppressed or induced by a chemical substance having a tamoxifen-like activity or the base sequence of EST How to screen.
[0039]
(39) A group C consisting of C1 to C11 genes including the following class 12 and class 16 genes or ESTs whose expression is suppressed or induced by a chemical substance having tamoxifen-like activity, and the class 8 and class 13 genes or ESTs: The method according to (38), wherein DNA comprising all or a part of one or more genes of D group consisting of D1 to D8 or a base sequence of EST is used.
[0040]
(Category 12) Competing with estrogen, no change with 5 μM tamoxifen, strong suppression with 10 nM tamoxifen alone
(C1) A1B1 (GenBank accession number AF137620) (SEQ ID NO: 23),
(C2) Estrogen receptor α (GenBank accession number AY033393) (SEQ ID NO: 24),
(C3) Homo sapiens UDP-Gal: betaGlcNAc beta 1,4-galactocyltransferase, polypeptide1 (GenBank accession number NM-001497) (SEQ ID NO: 25),
(C4) stearoyl-CoA desaturase (GenBank accession number XM-005719) (SEQ ID NO: 26),
(Category 16) Induced somewhat by 5μM tamoxifen due to competition with estrogen and strongly suppressed only by 10nM tamoxifen
(C5) cadherin 18 (GenBank accession number NM-004934) (SEQ ID NO: 27),
(C6) enolase 2 (GenBank accession number X51956) (SEQ ID NO: 28),
(C7) enolase 3 (GenBank accession number X56832) (SEQ ID NO: 29),
(C8) Human mRNA for KIAA0373 gene (GenBank accession number AB002371) (SEQ ID NO: 30),
(C9) Homo sapiens tumor-associated calcium signal transducer 2 (GenBank accession number XM-001710) (SEQ ID NO: 31),
(C10) Homo sapiens enolase 1 (alpha) (ENO1) (GenBank accession number NM-001428) (SEQ ID NO: 32), and
(C11) selenium binding protein 1 (GenBank accession number U29091) (SEQ ID NO: 33),
(Category 8) Competing with estrogen, no change with 5 μM tamoxifen, strongly induced with 10 nM tamoxifen alone
(D1) CDC6 homolog (GenBank accession number NM-001254) (SEQ ID NO: 34),
(D2) chromobox homolog 1 (GenBank accession number BC002609) (SEQ ID NO: 35),
(D3) Homo sapiens interleukin 2 receptor, beta (GenBank accession number XM-009962) (SEQ ID NO: 36),
(D4) Human mRNA for KIAA0008 (GenBank accession number D13633) (SEQ ID NO: 37),
(D5) Human mRNA for KIAA0101 gene (GenBank accession number D14657) (SEQ ID NO: 38),
(D6) KIAA1051 protein (GenBank Accession No. AB028974) (SEQ ID NO: 39)
(Category 13) Slightly induced by 5 μM tamoxifen due to competition with estrogen, but strongly induced only by 10 nM tamoxifen
(D7) Homo sapiens paired basic amino acid cleaving system 4 (GenBank accession number NM-002570) (SEQ ID NO: 40), and
(D8) neuropeptide Y receptor Y1 (GenBank accession number NM-000909) (SEQ ID NO: 41)
[0041]
(40) A DNA comprising all or part of one or more genes of C group consisting of C1-C11 gene or EST of (39) and one or more genes of D group consisting of D1-D8 gene or EST or EST base sequence (39) The method of (39) including analyzing the expression of this gene using the spotted DNA microarray.
(41) A DNA comprising all or part of one or more genes of C group consisting of C1-C11 gene or EST of (39) and one or more genes of D group consisting of D1-D8 gene or EST, or EST base sequence The method according to (39), comprising analyzing expression of the gene by targeted real-time PCR.
[0042]
(42) A DNA comprising all or part of one or more genes of C group consisting of C1-C11 gene or EST of (39) and one or more genes of D group consisting of D1-D8 gene or EST or EST base sequence A DNA microarray for screening a spotted substance that acts in the same manner as tamoxifen on the expression of the gene or EST.
(43) A substance that acts in the same manner as tamoxifen on the expression of the gene or EST, using a DNA containing all or part of the base sequence of the gene or EST whose expression is affected by a chemical substance having tamoxifen-like activity How to screen.
[0043]
(44) The group A consisting of the genes A1 to A15 or EST of (24) and the group B consisting of the genes or ESTs of B1 to B7 and the group C consisting of the genes C1 to C11 or EST of (39) and D1 to D8 The method according to (43), wherein one or more genes of the D group consisting of the gene or EST or DNA containing all or part of the base sequence of the EST is used.
(45) The group A consisting of the genes A1 to A15 or EST of (24) and the group B consisting of the genes or ESTs of B1 to B7 and the group C of the genes C1 to C11 or EST of (39) and D1 to D8 A substance that acts in the same manner as tamoxifen on the expression of the gene or EST, by spotting DNA containing all or part of the EST base sequence of one or more genes of the D group consisting of the gene or EST DNA microarray for.
Hereinafter, the present invention will be described in detail.
[0044]
DETAILED DESCRIPTION OF THE INVENTION
1. Acquisition of genes that can be used in the method of the present invention
(1) Among genes whose expression varies depending on a chemical substance having an estrogen-like activity, a gene whose expression is different between when stimulated by both estrogen and tamoxifen and when only a chemical substance having an estrogen-like activity exists Or get EST
The gene of the present invention can be selected from genes whose expression is affected by stimulation with a chemical substance having estrogen-like activity. As used herein, estrogen refers to 17β estradiol, and examples of chemical substances having estrogen-like activity include estrone, estriol, diethylstilbestrol (DES), bisphenol A, and nonylphenol.
[0045]
Selection of a gene can be performed by stimulating an estrogen-responsive cell with estrogen and comparing the gene expression profile of the cell with that of the cell not stimulated with estrogen. Here, examples of the cells to be used include human breast cancer cells and intrauterine tumor cells. For example, human breast cancer cells MCF-7 can be used. Stimulation with estrogen can be performed, for example, by culturing cells in an appropriate medium, adding estrogen to the cultured cells at a concentration of several nM to several tens of μM, and further culturing for several days.
[0046]
Expression profiles include, for example, a method using a DNA chip, a method using a DNA microarray, a cDNA subtraction method (cDNA subtraction), a differential display method (differential display), SAGE (serial analysis of gene expression), ATAC-PCR, Taq Man It can be performed by real-time PCR such as PCR. The gene selection method of the present invention is preferably performed using a DNA chip or a DNA microarray in terms of comprehensive expression analysis. A DNA chip can be prepared by spotting genetic DNA or EST of which the sequence is known in advance using a pin head or an ink jet on a substrate, or by synthesizing on a suitable substrate by optical lithography. At this time, DNA sequence information can be obtained from a DNA database such as GenBank. A DNA microarray can be prepared by obtaining a cDNA clone from a human cell, amplifying the cDNA by PCR, and affixing it on a suitable substrate.
Analysis by these methods should be performed according to the description of “DNA microarray and latest PCR method”, separate volume of cell engineering, Genome Science Series 1, supervised by Masaaki Muramatsu, Hiroyuki Nami, published on March 16, 2000, Shujunsha etc. Can do.
[0047]
Here, the DNA microarray is a generic term for devices in which DNA is aligned (arrayed) and immobilized on a substrate. Depending on the size of the substrate and the density of the DNA to be loaded, it can be divided into DNA macroarrays and DNA microarrays. In the present invention, both DNA macroarrays and DNA microarrays are called DNA microarrays. Further, the number of DNAs to be aligned is not limited, and is included in the DNA microarray as long as at least one DNA is spotted on the substrate. Furthermore, in a DNA microarray, DNA spotted on a substrate is hybridized with a labeled sample DNA, but this labeling method is not limited, and includes fluorescent labeling, labeling with a radioisotope, and the like.
[0048]
For example, the gene of the present invention can be selected using a cDNA microarray as follows. First, human EST or full-length cDNA is obtained. A cDNA library can be prepared from human cells, or a commercially available gene set may be used. The obtained cDNA and EST are used as probe DNA and spotted on a glass slide using a commercially available array machine. As the slide glass to be used, for example, a glass treated with poly-L-lysine is used. For example, DNA used for immobilization is dissolved in 3 × SSC and spotted. The concentration is not limited, but a concentration of 0.5 μg / μl to 1 μg / μl or more is desirable. For example 4cm ² In the case of glass slides, it is possible to immobilize 10000 or more kinds of DNA per sheet by spotting at a diameter of about 100 to 400 μm. After immobilization, post-treatment is performed by rehydration, blocking with succinic acid, and the like.
[0049]
Subsequently, mRNA is extracted from cells stimulated with estrogen and cells not stimulated with estrogen, and labeled cDNA is prepared from the mRNA. mRNA is prepared by extracting total RNA by the guanidine thiocyanate / cesium chloride method, etc., and then using an affinity column method such as oligo dT-cellulose or poly U-sepharose, or a batch method to obtain poly (A) + RNA (mRNA ). The cDNA may be labeled when a single-stranded cDNA is synthesized using the thus obtained mRNA as a template using an oligo dT primer and reverse transcriptase. Labeling may be performed with a fluorescent dye, and a cDNA obtained from cells stimulated with estrogen and a cDNA obtained from cells not stimulated with estrogen may be labeled with different fluorescent dyes. Examples of fluorescent dyes include Cy3 (red) and Cy5 (green) from Amersham Pharmacia Biotech.
[0050]
CDNA labeled with each fluorescent dye in a suitable hybridization solution is hybridized with the DNA spotted on the microarray. The hybridization conditions at this time can be appropriately selected by changing the salt concentration of the hybridization solution or changing the reaction temperature according to the desired sensitivity.
[0051]
After hybridization, by observing or measuring the fluorescence pattern on the slide glass with a fluorescence microscope, fluorescence detector (eg, CHIP READER ™ (manufactured by VERTEK)), etc., do not stimulate with estrogen-stimulated cells and estrogen The expression profile of the cells can be analyzed. For example, if cDNA obtained from cells stimulated with estrogen is labeled with red Cy3 and cDNA obtained from cells not stimulated with estrogen is labeled with green Cy5, a gene that is expressed only in cells stimulated with estrogen The spot on the immobilized microarray emits red fluorescence, and the spot on the microarray to which genes expressed only in cells that are not stimulated with estrogen emit green fluorescence, on the microarray on which the genes expressed in both cells are immobilized. The spot emits a composite color fluorescence of red and green. DNA immobilized on a spot that shows a result that Cy3 fluorescence (red fluorescence) intensity when stimulated with estrogen is greater than Cy5 fluorescence (green fluorescence) intensity when not stimulated with estrogen is a gene whose expression is increased by estrogen stimulation. Yes, DNA immobilized on a spot showing a result that the fluorescence intensity of Cy3 when stimulated with estrogen is smaller than the fluorescence intensity of Cy5 when not stimulated with estrogen is a gene whose expression is suppressed by estrogen stimulation. The fluorescence intensity of Cy3 and the fluorescence intensity of Cy5 at each spot on the DNA microarray can be measured, for example, the Cy3 / Cy5 ratio can be calculated, and the effect of estrogen on these genes or ESTs can be evaluated using the ratio as an index. At this time, an internal standard gene is spotted on the DNA microarray. DNA that can hybridize with the internal standard gene and labeled with Cy3 or Cy5 is added to the sample for hybridization. When calculating the Cy3 / Cy5 ratio on each spot, the Cy3 / Cy5 ratio of each spot can be corrected by normalizing with the Cy3 / Cy5 ratio of the internal standard gene.
[0052]
Selected genes and ESTs can be classified according to expression profiles as follows.
Group A: The combination of estrogen and tamoxifen suppresses the expression level compared to when only estrogen is present.
Group B: A combination of estrogen and tamoxifen induces more expression than when only estrogen is present.
[0053]
Furthermore, by changing the concentrations of estrogen and tamoxifen used for cell stimulation, the genes and ESTs of Group A and Group B can be further subdivided according to the concentration of each substance required for expression variation.
For example, the following can be mentioned as genes of group A or EST, and further subclassified as follows depending on whether the expression is suppressed by 5 μM and 10 nM tamoxifen. This indicates that the degree of tamoxifen competitively suppressing the effect of estrogen on the gene and the degree of influence of tamoxifen on the gene differ depending on the gene. The accession number of GenBank is shown in parentheses after the following gene or EST name. In addition, since the gene name (Definition) may be changed on the gene database, as long as the gene has the accession number described in this specification, even if the name on the database is changed, the present invention Included in genes used for
[0054]
(Category 1) Strongly suppressed by 5μM tamoxifen due to competition with estrogen, and slightly induced only by 10nM tamoxifen.
Homo sapiens methylene tetrahydrofolate dehydrogenase (XM-002234)
[0055]
(Category 2) Strongly suppressed by 5μM tamoxifen due to competition with estrogen, but no change with 10nM tamoxifen alone.
asparagine synthetase (L35946)
EST (AA587912)
exportin (AF039022)
ferritin, heavy polypeptide 1 (AA102267)
glutamine-fructose-6-phosphate transaminase 1 (M90516)
78KD GLUCOSE REGULATED PROTEIN PRECURSOR (AI878886)
Homo sapiens partial mRNA for A-type potassium channel modulatory protein 2 (AJ276317)
insulin-like growth factor-binding protein 4 (M62403)
methylene tetrahydrofolate dehydrogenase (X16396)
phosphoenolpyruvate carboxykinase 2 (X92720)
protein kinase C, delta (D10495)
trefoil factor 1 (X05030)
[0056]
(Category 3) Those that are strongly suppressed by 5 μM tamoxifen due to competition with estrogen and slightly suppressed only by 10 nM tamoxifen.
argininosuccinate synthetase (AI660571)
solute carrier family 7, member 5 (XM-017410)
Similarly, the following can be mentioned for the genes of group B.
[0057]
(Category 17) Strongly induced by 5 μM tamoxifen in competition with estrogen, but not changed by 10 nM tamoxifen alone.
erbB2 (M86910)
fucosyltransferase 8 (Y17977)
Homo sapiens, adipose specific 2 (BC004471)
insulin-like growth factor binding protein 5 (L27556)
[0058]
(Category 18) Strongly induced by 5 μM tamoxifen in competition with estrogen and slightly suppressed by 10 nM tamoxifen alone.
Homo sapiens DNA from chromosome 19 (AC004030)
quiescin Q6 (U97276)
(Category 19) Strongly induced by 5 μM tamoxifen in competition with estrogen and strongly suppressed by 10 nM tamoxifen alone.
solute carrier family 12, member 2 (XM-003745)
[0059]
In this way, the obtained genes or ESTs whose expression is varied by estrogen stimulation are spotted on a slide glass to prepare a DNA microarray.
In the present invention, the above gene or EST includes not only the above gene or EST but also DNA that hybridizes under stringent conditions with a DNA having a base sequence complementary to the base sequence of the above gene or EST. . Here, stringent conditions refer to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, DNAs having high homology, that is, DNAs having a homology of 60% or more, preferably 80% or more hybridize, whereby nucleic acids having low homology do not hybridize. More specifically, the sodium concentration is 150 to 900 mM, preferably 600 to 900 mM, and the temperature is 60 to 68 ° C, preferably 65 ° C.
[0060]
Furthermore, the full length of the above-mentioned gene or EST may be used, or a partial fragment may be used. When using a partial fragment, the position of the partial sequence is not limited, and a continuous partial sequence of each gene or EST may be used. The length of the fragment is not limited.
In addition, as a DNA microarray in which these genes and EST are immobilized, for example, InfoArray (trademark) manufactured by InfoJeans can be used.
[0061]
The above-mentioned cells are stimulated with one or both of estrogen and tamoxifen, and an expression profile with cells that are not stimulated was obtained by the above method. By comparing using `` of genes whose expression varies due to chemical substances having estrogen-like activity, when stimulated by both estrogen and tamoxifen and when only chemical substances having estrogen-like activity exist, Genes or ESTs with different expression can be obtained. Stimulation with estrogen and tamoxifen can be performed by culturing the cells for several days in a medium in which both substances are mixed at appropriate concentrations.
[0062]
(2) Acquisition of genes or ESTs whose expression varies with chemical substances having tamoxifen-like activity
1. In the method of obtaining a gene or EST whose expression varies depending on a chemical substance having an estrogen-like activity in (1), by using a chemical substance having a tamoxifen-like activity instead of estrogen, “by a chemical substance having a tamoxifen-like activity” Genes or ESTs whose expression varies can be obtained. In addition, By using the DNA microarray in which the gene whose expression is changed by the chemical substance having the estrogen-like activity obtained in (1) is immobilized, among the genes whose expression is changed by the chemical substance having the estrogen-like activity, the expression is expressed by tamoxifen. Fluctuating genes or ESTs can be obtained. Here, examples of the chemical substance having tamoxifen-like activity include tamoxifen, triphenylethylene, clomiphene, toremifine, ICI182,780 and the like. However, since many estrogen antagonists including tamoxifen have not only an estrogen activity inhibitory effect but also various degrees of agonist activity, is the gene or EST whose expression changed by tamoxifen due to the antagonistic effect of tamoxifen? Or it is difficult to describe exactly whether it is due to an agonistic effect. Therefore, it is necessary to evaluate the effect by whether or not the variation shown by the gene or EST shown by giving the test substance is similar to the change shown by tamoxifen, or by the degree of similarity.
[0063]
Genes or ESTs whose expression varies depending on chemical substances having tamoxifen-like activity can be classified as follows.
Group C: those whose expression level is suppressed by the presence of tamoxifen.
Group D: expression level is induced by the presence of tamoxifen.
[0064]
Furthermore, by changing the concentrations of estrogen and tamoxifen used for cell stimulation, the genes and ESTs of Group C and Group D can be further subdivided according to the concentration of each substance required for expression fluctuation.
For example, group C genes or ESTs include the following, and whether expression is suppressed or induced when stimulated simultaneously with estrogen and tamoxifen (5 μM) and when stimulated with tamoxifen alone (10 nM): Then, it is subdivided as follows. This indicates that the degree of tamoxifen competitively suppressing the effect of estrogen on the gene and the degree of influence of tamoxifen on the gene differ depending on the gene. The accession number of GenBank is shown in parentheses after the following gene or EST name.
[0065]
(Category 12) Competing with estrogen, no change with 5 μM tamoxifen, strong suppression with 10 nM tamoxifen alone.
A1B1 (AF137620)
Estrogen receptor α (AY033393)
Homo sapiens UDP-Gal: betaGlcNAc beta 1,4-galactocyltransferase, polypeptide1 (NM-001497)
stearoyl-CoA desaturase (XM-005719)
[0066]
(Category 16) Slightly induced by 5 μM tamoxifen due to competition with estrogen and strongly suppressed by 10 nM tamoxifen alone.
cadherin 18 (NM-004934)
enolase 2 (X51956)
enolase 3 (X56832)
Human mRNA for KIAA0373 gene (AB002371)
Homo sapiens tumor-associated calcium signal transducer 2 (XM-001710)
Homo sapiens enolase 1 (alpha) (ENO1) (NM-001428)
selenium binding protein 1 (U29091)
Similarly, the following are mentioned as a gene or EST of D group.
[0067]
(Category 8) Competing with estrogen, no change with 5 μM tamoxifen, strong induction with 10 nM tamoxifen alone.
CDC6 homolog (NM-001254)
chromobox homolog 1 (BC002609)
Homo sapiens interleukin 2 receptor, beta (XM-009962)
Human mRNA for KIAA0008 (D13633)
Human mRNA for KIAA0101 gene (D14657)
KIAA1051 protein (AB028974)
[0068]
(Category 13) Slightly induced by 5 μM tamoxifen in competition with estrogen and strongly induced only by 10 nM tamoxifen.
Homo sapiens paired basic amino acid cleaving system 4 (NM-002570)
neuropeptide Y receptor Y1 (NM-000909)
[0069]
In this way, the obtained gene or EST is immobilized on a slide glass to prepare a DNA microarray.
In the present invention, the above gene or EST includes not only the above gene or EST but also a DNA that hybridizes under stringent conditions with a DNA having a base sequence complementary to the base sequence of the above gene or EST. . Here, stringent conditions refer to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, DNAs having high homology, that is, DNAs having a homology of 60% or more, preferably 80% or more hybridize, whereby nucleic acids having low homology do not hybridize. More specifically, the sodium concentration is 150 to 900 mM, preferably 600 to 900 mM, and the temperature is 60 to 68 ° C, preferably 65 ° C.
[0070]
2. DNA microarray for evaluating tamoxifen's estrogenic activity inhibitory effect, tamoxifen effect, or tamoxifen-like effect of test substances
By immobilizing the above-mentioned gene and the full-length sequence or partial sequence of EST on an appropriate substrate, it can be used to evaluate the effect of tamoxifen on suppressing estrogen activity, the effect of tamoxifen, or the effect of tamoxifen on the test substance DNA microarrays can be prepared.
[0071]
Examples of the fixed substrate include a glass plate, a quartz plate, and a silicon wafer. Examples of the substrate size include 3.5 mm x 5.5 mm, 18 mm x 18 mm, and 22 mm x 75 mm, but this can be set according to the number of DNA probe spots on the substrate and the size of the spots. can do. As a DNA immobilization method, an appropriate method is selected according to the type of DNA and the type of carrier. For example, when the DNA to be immobilized is a cDNA or PCR product, it can be electrostatically bound to a solid support surface treated with a polycation such as polylysine, polyethyleneimine, or polyalkylamine using the charge of the DNA. Immobilization in which DNA introduced with a functional group such as amino group, aldehyde group, SH group or biotin can be covalently bound to a solid phase surface introduced with a functional group such as an aldehyde group, an epoxy group, What is necessary is just to perform using an array machine as mentioned above. In this case, the full length may be used as the gene or EST to be immobilized, or a partial fragment may be used. When using a partial fragment, the position of the partial sequence is not limited, and a continuous partial sequence of each gene or EST may be used. The length of the fragment is not limited. What is necessary is just to fix | immobilize the full length DNA of the gene of A group-D group, or its fragment | piece. All the genes or ESTs of groups A to D may be immobilized on one slide glass, or may be immobilized for each group, or at least one gene or EST is selected from each group and immobilized. May be.
[0072]
3. A method for evaluating the effect of tamoxifen on estrogen activity, the effect of tamoxifen, or the tamoxifen-like effect of a test substance.
(1) Among genes whose expression varies depending on a chemical substance having an estrogen-like activity, a gene whose expression is different between when stimulated by both estrogen and tamoxifen and when only a chemical substance having an estrogen-like activity exists Or EST, (2) among genes whose expression varies due to a chemical substance having an estrogen-like activity, or a gene or EST whose expression rebounds when stimulated with tamoxifen, and (3) a chemical substance having a tamoxifen-like activity The gene or EST whose expression varies can be used to evaluate the effect of tamoxifen on the estrogen activity, the effect of tamoxifen, or the effect similar to that of a test substance whose action is unknown.
[0073]
Here, in order to screen for an unknown estrogen activity inhibitor, tamoxifen should be based on the estrogen activity inhibitory effect. This is because an unknown substance that exhibits an estrogen activity inhibitory effect by the same mechanism as that exhibited by tamoxifen is expected to show the same gene response as tamoxifen, and is unknown by examining the response of a specific group of genes. This means that the same effect as tamoxifen can be evaluated. That is, by evaluating the inhibitory effect of tamoxifen on estrogen activity based on the expression of many estrogen-responsive genes, and comparing the effects of unknown substances on the expression of the genes with the results obtained with tamoxifen. The effect of unknown substances can be evaluated. In the present invention, evaluating the effect of tamoxifen on the estrogenic activity and the effect of tamoxifen based on gene expression is meaningful in that the effect of tamoxifen can be used as a reference for evaluating the effects of unknown substances.
[0074]
Here, “evaluation of the inhibitory effect of tamoxifen on estrogen activity” refers to which gene or EST expression of tamoxifen is analyzed by analyzing the expression of the gene or EST of (1) above when stimulated with estrogen and tamoxifen. Determining whether to suppress or induce the degree to suppress estrogen activity. “Evaluation of the effects of tamoxifen” refers to the suppression of which gene or EST expression of tamoxifen by analyzing the expression of the gene (2) or (3) or EST when stimulated with tamoxifen. Or to decide whether to induce. The “evaluation of the tamoxifen-like (tamoxifen-like) effect of the test substance” means that the expression of the gene or EST of (1) above when stimulated with estrogen and the test substance is analyzed, and the test substance is the gene or EST A method of determining whether or not a test substance has an effect of inducing the gene or EST to respond to estrogen in the same manner as tamoxifen, Thus, a substance having an action similar to that of tamoxifen can be screened by suppressing or inducing the response between the estrogen gene and estrogen. Furthermore, “evaluation of the tamoxifen-like (tamoxifen-like) effect of the test substance” means that the test substance is analyzed by analyzing the expression of the gene (2) or (3) or EST when stimulated with the test substance. It is also possible to determine whether the expression of the gene or EST is suppressed or induced in the same manner as tamoxifen, and by directly or indirectly acting on the estrogen responsive gene by the method, screening for a substance having an action similar to tamoxifen Can do.
[0075]
For example, by using the above-mentioned group A genes or ESTs, it is possible to evaluate whether or not the test substance suppressed tamoxifen-like suppression of these genes or ESTs in response to estrogen. It can also be evaluated whether the tamoxifen-like substance behaved the same as tamoxifen. In addition, by using the genes or ESTs of group B, it is possible to evaluate whether or not the expression level of the test substance in these genes or ESTs was induced like tamoxifen in cooperation with estrogen. It can also be evaluated whether the tamoxifen-like substance behaved the same as tamoxifen. In addition, by using genes or ESTs of group C, it is possible to evaluate whether the test substance suppresses the expression level of these genes or ESTs like tamoxifen. It can also be evaluated whether the tamoxifen-like substance behaved the same as tamoxifen. Furthermore, by using the genes or ESTs of group D, it is possible to evaluate whether the test substance induces the expression level of these genes or ESTs like tamoxifen. It can also be evaluated whether the tamoxifen-like substance behaved the same as tamoxifen.
[0076]
Therefore, tamoxifen alone, tamoxifen and estrogen, and the test substance and estrogen stimulated with estrogen, by examining the expression of each gene for each group, tamoxifen estrogen activity inhibitory effect, tamoxifen effect or action tamoxifen of unknown test substance Similar effects can be evaluated. However, it is difficult to strictly distinguish whether the variation in gene expression in each group is due to the effect of suppressing the estrogen activity of tamoxifen or the test substance or the influence of tamoxifen or the test substance on its own gene. It is preferable to examine the expression of these genes. However, genes can be classified according to their functions, but each function is completely interchangeable between genes, is not compatible despite showing similar functions, or is not compatible because the functions are completely different. Furthermore, even if they are interchangeable, the efficiency (activity) of the function may differ, and it is currently impossible to clearly describe the function and activity. Therefore, it is preferable not to classify by function or activity type, but to classify by the degree of gene expression response to the drug. In particular, regarding the estrogen activity inhibitory effect, it is thought to be due to competitive action against estrogen and the structure of each drug. It is preferable to classify based on the effect on gene expression.
[0077]
1. The genes and ESTs of groups A to D selected by the method of (1) are genes whose expression is affected by estrogen and tamoxifen. Both the expression profile and the estrogen and test substance when cultured cells are stimulated with estrogen alone By analyzing the expression profile when stimulated with, it is possible to determine whether or not the test substance has an effect of suppressing estrogen activity in the same manner as tamoxifen. For example, if a gene or EST that increases in expression when stimulated only with estrogen does not increase in expression when stimulated with both estrogen and the test substance, the test substance is evaluated as having an estrogen activity inhibitory effect can do.
[0078]
Furthermore, as described above, the genes in groups A to D are suppressed or induced when further stimulated with estrogen and tamoxifen (5 μM) and stimulated with tamoxifen alone (10 nM). It is subdivided as described above. This indicates that the degree of tamoxifen competitively suppressing the effect of estrogen on the gene and the degree of influence of tamoxifen on the gene differ depending on the gene. Each test substance is classified into sub-classification groups (class A, group 1, 2, 3 for group A, classes 17, 18, 19 for group B, classes 12 and 16 for group C, classes 8 and 13 for group D) By examining whether to suppress or induce the expression of a gene or EST, the influence of suppression of estrogen activity other than suppression of estrogen activity of tamoxifen can be eliminated, and screening of a test substance can be performed more precisely. The genes in groups A to D described above are classified based on gene responses when stimulated simultaneously with estrogen and tamoxifen (5 μM) and when stimulated with tamoxifen alone (10 nM). As a basis for this, 10 nM estrogen is the reference concentration for evaluating the estrogen effect, and the concentration of tamoxifen that exhibits competitive inhibitory activity is about 5 μM. Therefore, this condition was used as a standard for competitive reactions. To see the unique effects of Tamoxifen, we used the same gene response at 10 nM as estrogen. These concentrations are an example and include ranges of estrogen and tamoxifen concentrations, respectively, in the range where similar gene function can be expected. The same applies to the concentration of the test substance.
[0079]
To evaluate the effects of tamoxifen on estrogen activity, the effects of tamoxifen, and the effects of tamoxifen on the test substance, the most accurate evaluation can be performed using all of the genes in Group A to Group D or EST. Alternatively, EST may be used. The following shows what kind of effect or substance can be screened by using which gene or EST in the case of evaluating the tamoxifen-like effect of the test substance.
[0080]
To screen for substances that suppress estrogen-responsive genes or EST responses to estrogens in the same way as tamoxifen, one or more genes belonging to Group A or ESTs may be used. More preferably, a total of 3 or more genes or ESTs, each one or more from the genes or ESTs belonging to 1, 2, 3), are used. For screening for substances that induce gene or EST response to estrogen in the same manner as tamoxifen, one or more genes belonging to group B or EST may be used, and subclassification of group B (classification 17, 18, It is more preferable to use a total of 3 or more genes or ESTs, each one or more from the genes or ESTs belonging to 19). To broadly screen for substances that inhibit or induce estrogen-responsive genes or ESTs to respond to estrogen, as well as tamoxifen, one or more genes in group A or EST and one or more genes in group B Use EST. At this time, a total of 6 or more genes or ESTs may be used for each subclassification of group A and each subclassification of group B and a total of 6 or more genes or ESTs.
[0081]
Like tamoxifen, estrogen-responsive genes or substances that suppress the expression of ESTs can be screened by using one or more genes belonging to group C or EST, and subclassification of group C (classifications 12 and 16). It is more preferable to use a total of two or more genes or ESTs, each of which is one or more from the genes or ESTs belonging to. Similar to tamoxifen, estrogen-responsive genes or substances that induce EST expression can be screened by using one or more genes belonging to group D or EST. Subclassification of group D (classification 8, 13) It is more preferable to use a total of two or more genes or ESTs, each of which is one or more from the genes or ESTs belonging to. By using one or more of the group C genes or ESTs and one or more of the group D genes or ESTs, substances having similar activity to tamoxifen can be widely screened. At this time, a total of four or more genes or ESTs may be used for each of the subclasses of group C and subclasses of group D and a total of four or more genes or ESTs.
[0082]
In addition, if you use one or more of group A genes or ESTs, one or more of group B genes or ESTs, one or more of group C genes or ESTs and one or more of group D genes or ESTs, A substance having an action similar to tamoxifen can be screened, and the action of the substance can be determined by the expression profile of the gene or EST used. In this case, one or more from each sub-class of Group A, one or more from each sub-class of Group B, one or more from each sub-class of Group C and one or more from each sub-class of Group D If a total of 10 or more genes or ESTs are used, screening can be performed more accurately.
[0083]
Among genes whose expression varies depending on chemical substances having estrogen-like activity, genes or ESTs whose expression is different between when stimulated with both estrogen and tamoxifen and when only chemical substances with estrogen-like activity exist By using the spotted DNA microarray, the effect of suppressing the estrogen activity of tamoxifen, the effect of tamoxifen or the effect similar to tamoxifen of a test substance whose action is unknown can be evaluated. It can also be evaluated by real-time PCR or the like targeting the gene or EST.
[0084]
For example, in the case of using a DNA microarray, the above-described gene, DNA, and DNA microarray on which an internal standard gene is immobilized are used as follows. The cells described above are stimulated with tamoxifen or the test substance and / or estrogen. Stimulation can be performed by culturing the cells in an appropriate medium, adding one or both of tamoxifen or the test substance and estrogen to the medium, and further culturing. The concentration to be added is not limited, but it may be added from several nM to several tens of μM. Next, mRNA is extracted from the cultured cells to produce cDNA labeled with a fluorescent dye. At this time, for example, a cDNA prepared from cells stimulated with both the test substance and estrogen is labeled with Cy3, and a cDNA prepared from cells stimulated only with estrogen is labeled with Cy5. By hybridizing the labeled cDNA with a microarray and measuring the fluorescence of Cy3 and Cy5 on each spot, the expression level of the gene or EST on each spot is known, and how much expression is suppressed or induced. And an expression profile can be obtained. By comparing the expression profile obtained with the test substance with the expression profile of tamoxifen, it can be determined whether the test substance has an effect similar to tamoxifen. Thus, the effect of tamoxifen or a test substance can be evaluated by analyzing the expression profile in cultured cells.
[0085]
Compared to stimulation with estrogen alone when stimulated with both the test substance and estrogen, the test substance has the same activity as tamoxifen when the expression of the above-mentioned group A gene or EST is suppressed. It can be evaluated that it is a substance to be suppressed. In addition, when the expression of the group B gene or EST is induced, the test substance can be evaluated as a substance that induces estrogen activity in the same manner as tamoxifen.
[0086]
Further, when the above-mentioned group C gene or EST expression is suppressed when stimulated with a test substance, the test substance can be evaluated as a substance exhibiting the same effect as tamoxifen, and the group D gene Alternatively, even when EST expression is induced, the test substance can be evaluated as a substance exhibiting the same effect as tamoxifen.
[0087]
The analysis by real-time PCR can be performed with an apparatus in which a thermal cycler and a spectrofluorometer are integrated, for example, LightCycler (trademark) manufactured by Roche. Real-time PCR makes it possible to detect and analyze the production process of PCR amplification products in real time. Since the PCR product is labeled with a fluorescent dye, accurate quantification can be performed, which is suitable for the method of the present invention.
[0088]
Real-time PCR can be performed using LightCycler (Roche Diagnostics) and a hot start reaction solution kit LightCycler-FastStart DNA Master SYBR Green I (Roche Diagnostics). The procedure using this kit is described below. First, the primer mixture and the template cDNA solution are heat-treated at 82 ° C. for 5 minutes and then rapidly cooled in ice. Next, H ₂ O 12.6 mL, MgCl ₂ Mix 2.4 mL, 10 × Light Cycler Mix 2.0 mL, and template cDNA 1.0 mL to make a mixed solution. Dispense 19 mL of the prepared mixed solution into the capillary, add 1 mL of the primer mixture (10 pmol / mL) to it, and cap the capillary. Centrifuge at 1900 rpm for 10-15 seconds with the capillary on the centrifuge adapter. Set this in the LightCycler carousel and perform real-time PCR. Perform the above operations on ice or on a centrifuge adapter that has been stored at 4 ° C. The analysis can be performed using LightCycler Software Ver.3.3 (Roche Diagnostics).
[0089]
【Example】
EXAMPLES The present invention will be specifically described below with reference to examples, but the scope of the present invention is not limited to these examples.
[Example 1]
Cell culture is performed as follows. First, MCF7 cells are cultured in a liquid medium (eg, GibcoBRL trade name RPMI Medium 1640 containing Sigma's Fetal Bovine Serum at a concentration of 10%). Furthermore, in order to eliminate the influence of estrogen (17β estradiol), it is cultured for 5 days in a liquid medium containing Fetal Bovine Serum treated with charcoal at a concentration of 10%. Next, the compound to be examined for effects (for example, ethanol in the control group; control, 10 nM estrogen (E2) +5 μM tamoxifen (TAM) in the experimental group) was added to each of the above cultured cells, followed by charal-treated Fetal. Incubate for 72 hours in a liquid medium containing 10% Bovine Serum.
[0090]
In order to prepare a probe from the control group and the experimental group, a total amount of mRNA is extracted from each cultured cell (for example, trade name MACS manufactured by Miltenyi Biotec). A cDNA sample is prepared from the extracted total mRNA with reverse transcriptase (eg, GibcoBRL, SuperScript Reverse Transcriptase) and labeled dNTP (eg, Amersham Pharmacia Biotech, Cy3-dUTP, Cy5-dUTP). For example, cDNA using the total amount of mRNA extracted from the control group as a template is labeled with Cy5-dUTP, and cDNA using the total amount of mRNA extracted from the experimental group as a template is labeled with Cy3-dUTP and used as a probe.
[0091]
The prepared probe was hybridized with InfoArray (a microarray in which 204 estrogen response genes or 27 ESTs including 27 internal standard genes per EST), and then the hybridized cDNA was detected by a detector ( For example, the fluorescence intensity of Cy3 and Cy5 is measured using a product name “CHIP READER” manufactured by VERTEK.
[0092]
Analyze the fluorescence intensity of Cy3 and Cy5 for quantification. Analysis is performed as follows. First, the average value of the Cy3 / Cy5 ratio of the internal standard gene is calculated (at this time, spots with very low fluorescence intensity are excluded from the calculation). Calculate the Cy3 / Cy5 ratio for each gene or EST spot (again, spots with very low fluorescence intensity are excluded from the calculation). The Cy3 / Cy5 ratio of each gene or EST spot is normalized with the average value of the Cy3 / Cy5 ratio of the internal standard gene.
[0093]
[Table 1]

[0094]
Description of Table 1
A complete list of genes or ESTs that were used to evaluate the effects of tamoxifen and the estrogen activity-suppressing effect of tamoxifen. Twenty-seven control genes or ESTs are indicated by shading.
[0095]
(Description of each column)
Gene Name: Name registered in the database (GenBank, EMBL, DDBJ). However, abbreviations and common names are also included.
Accession No .: Accession number in the above database.
10 nM E2 Raw DATA: Value when 10 nM estrogen is allowed to act on MCF7 cells.
10 nM E2 + 5 μM TAM Raw DATA: Value obtained when 10 nM estrogen and 5 μM tamoxifen (TAM) were simultaneously applied to MCF7 cells.
10 nM E2 / 10 nM E2 + 5 μM TAM: The quotient of the value when 10 nM estrogen is allowed to act on MCF7 cells and the value when 10 nM estrogen and 5 μM tamoxifen (TAM) are allowed to act simultaneously on MCF7 cells.
10 nM TAM: Value when only 10 nM tamoxifen (TAM) is allowed to act on MCF7 cells.
[0096]
[Table 2]

[0097]
Description of Table 2
In Table 1, each gene or EST was classified into 19 items according to the following criteria. 1) In Table 1, the value of (10nM E2 / 10nM E2 + 5μM TAM) is 3.00 or higher, that is, the result of causing estrogen 10nM and tamoxifen 5μM to compete with each other and acting on MCF-7 cells, is strongly suppressed by tamoxifen 5μM The value of (10nM E2 / 10nM E2 + 5μM TAM) is 2.99 or less 1.50 or more, that is, slightly suppressed, the value of (10nM E2 / 10nM E2 + 5μM TAM) is 1.49 or less, 1.49 or more The value of (10nM E2 / 10nM E2 + 5μM TAM) is -1.50 or less -1.90 or more, that is, slightly induced, (10nM E2 / 10nM E2 + 5μM TAM) Is less than -2.00, ie strongly induced.
[0098]
1) Each of the items classified above was further classified according to the following criteria from the result of acting only 10 nM tamoxifen on MCF-7 cells. In Table 1, the value of (10nM TAM) is -2.00 or less, that is, strongly suppressed by tamoxifen 10nM, the value of (10nM TAM) is 1.90 or more and -1.50 or less, that is, slightly suppressed by tamoxifen 10nM A value of (10 nM TAM) of 1.49 or more and 1.49 or less, that is, no change by tamoxifen 10 nM, a value of (10 nM TAM) of 1.50 or more and 1.99 or less, that is, a product slightly induced by tamoxifen 10 nM, (10 nM TAM) value of 2.00 or higher, that is, strongly induced by tamoxifen 10 nM. As a result of classification according to this standard, a total of 19 items were classified.
[0099]
【The invention's effect】
As shown in the Examples, the gene or EST of the present invention exhibits a specific expression pattern when stimulated with estrogen or tamoxifen. The test substance suppresses estrogen activity or has tamoxifen-like activity by examining whether the test substance suppresses or induces the expression of these genes or ESTs in the presence or absence of estrogen Or not.
[0100]
[Sequence Listing]

Claims (12)

A DNA microarray in which DNAs containing all or part of the base sequences of the following four groups of genes from Group A to Group D or all EST genes or EST base sequences are spotted.
(1) Among the genes or ESTs whose expression is affected by a chemical substance having an estrogen-like activity, the expression is suppressed compared to the case where only a chemical substance having an estrogen-like activity is present when estrogens and tamoxifen are mixed. A group consisting of A1 to A15 containing the following class 1, class 2, and class 3 genes or ESTs that are induced or derived:
(Category 1) Strongly suppressed by 5 μM tamoxifen due to competition with estrogen, but slightly induced only by 10 nM tamoxifen
(A1) Homo sapiens methylene tetrahydrofolate dehydrogenase (XM-002234) (SEQ ID NO: 1),
(Category 2) 5 μM tamoxifen is strongly suppressed by competition with estrogen, 10 nM tamoxifen alone has no change,
(A2) asparagine synthetase (GenBank accession number L35946) (SEQ ID NO: 2),
(A3) EST (GenBank accession number AA587912) (SEQ ID NO: 3),
(A4) exportin (GenBank accession number AF039022) (SEQ ID NO: 4),
(A5) ferritin, heavy polypeptide 1 (GenBank accession number AA102267) (SEQ ID NO: 5),
(A6) glutamine-fructose-6-phosphate transaminase 1 (GenBank accession number M90516) (SEQ ID NO: 6),
(A7) 78KD GLUCOSE REGULATED PROTEIN PRECURSOR (GenBank accession number AI878886) (SEQ ID NO: 7),
(A8) Homo sapiens partial mRNA for A-type potassium channel modulatory protein 2 (GenBank accession number AJ276317) (SEQ ID NO: 8),
(A9) insulin-like growth factor-binding protein 4 (GenBank accession number M62403) (SEQ ID NO: 9),
(A10) methylene tetrahydrofolate dehydrogenase (GenBank accession number X16396) (SEQ ID NO: 10),
(A11) phosphoenolpyruvate carboxykinase 2 (GenBank accession number X92720) (SEQ ID NO: 11),
(A12) protein kinase C, delta (GenBank accession number D10495) (SEQ ID NO: 12),
(A13) trefoil factor 1 (GenBank accession number X05030) (SEQ ID NO: 13),
(Category 3) Strongly suppressed by 5 μM tamoxifen due to competition with estrogen, but slightly suppressed only by 10 nM tamoxifen
(A14) argininosuccinate synthetase (GenBank accession number AI660571) (SEQ ID NO: 14),
(A15) solute carrier family 7, member 5 (GenBank accession number XM-017410) (SEQ ID NO: 15),
(2) Among the genes or ESTs whose expression is affected by a chemical substance having an estrogen-like activity, the expression is suppressed compared to the case where only a chemical substance having an estrogen-like activity exists when estrogens and tamoxifen are mixed. Group B consisting of B1 to B7 containing the following class 17, class 18 and class 19 genes or ESTs derived or derived:
(Category 17) Strongly induced by 5 μM tamoxifen in competition with estrogen, with no change with 10 nM tamoxifen alone
(B1) erbB2 (GenBank accession number M86910) (SEQ ID NO: 16),
(B2) fucosyltransferase 8 (GenBank accession number Y17977) (SEQ ID NO: 17),
(B3) Homo sapiens, adipose specific 2 (GenBank accession number BC004471) (SEQ ID NO: 18),
(B4) insulin-like growth factor binding protein 5 (GenBank accession number L27556) (SEQ ID NO: 19),
(Category 18) Strongly induced by 5 μM tamoxifen due to competition with estrogen and slightly suppressed by 10 nM tamoxifen alone
(B5) Homo sapiens DNA from chromosome 19 (GenBank accession number AC004030) (SEQ ID NO: 20),
(B6) quiescin Q6 (GenBank accession number U97276) (SEQ ID NO: 21), and (Category 19) that is strongly induced by 5 μM tamoxifen in competition with estrogen and strongly suppressed only by 10 nM tamoxifen,
(B7) solute carrier family 12, member 2 (GenBank accession number XM-003745) (SEQ ID NO: 22),
(3) Group C consisting of C1 to C11 including the following class 12 and class 16 genes or ESTs whose expression is affected by a chemical substance having tamoxifen-like activity:
(Category 12) Competing with estrogen, no change with 5 μM tamoxifen, strong suppression with 10 nM tamoxifen alone
(C1) A1B1 (GenBank accession number AF137620) (SEQ ID NO: 23),
(C2) Estrogen receptor α (GenBank accession number AY033393) (SEQ ID NO: 24),
(C3) Homo sapiens UDP-Gal: betaGlcNAc beta 1,4-galactocyltransferase, polypeptide1 (GenBank accession number NM-001497) (SEQ ID NO: 25),
(C4) stearoyl-CoA desaturase (GenBank accession number XM-005719) (SEQ ID NO: 26),
(Category 16) Induced somewhat by 5μM tamoxifen due to competition with estrogen and strongly suppressed only by 10nM tamoxifen
(C5) cadherin 18 (GenBank accession number NM-004934) (SEQ ID NO: 27),
(C6) enolase 2 (GenBank accession number X51956) (SEQ ID NO: 28),
(C7) enolase 3 (GenBank accession number X56832) (SEQ ID NO: 29),
(C8) Human mRNA for KIAA0373 gene (GenBank accession number AB002371) (SEQ ID NO: 30),
(C9) Homo sapiens tumor-associated calcium signal transducer 2 (GenBank accession number XM-001710) (SEQ ID NO: 31),
(C10) Homo sapiens enolase 1 (alpha) (ENO1) (GenBank accession number NM-001428) (SEQ ID NO: 32),
(C11) selenium binding protein 1 (GenBank accession number U29091) (SEQ ID NO: 33),
(4) Group D consisting of D1 to D8 containing the genes or ESTs of the following class 8 and class 13 whose expression is affected by a chemical substance having tamoxifen-like activity:
(Category 8) Competing with estrogen, no change with 5 μM tamoxifen, strongly induced with 10 nM tamoxifen alone
(D1) CDC6 homolog (GenBank accession number NM-001254) (SEQ ID NO: 34),
(D2) chromobox homolog 1 (GenBank accession number BC002609) (SEQ ID NO: 35),
(D3) Homo sapiens interleukin 2 receptor, beta (GenBank accession number XM-009962) (SEQ ID NO: 36),
(D4) Human mRNA for KIAA0008 (GenBank accession number D13633) (SEQ ID NO: 37),
(D5) Human mRNA for KIAA0101 gene (GenBank accession number D14657) (SEQ ID NO: 38),
(D6) KIAA1051 protein (GenBank accession number AB028974) (SEQ ID NO: 39),
(Category 13) Slightly induced by 5 μM tamoxifen due to competition with estrogen, but strongly induced only by 10 nM tamoxifen
(D7) Homo sapiens paired basic amino acid cleaving system 4 (GenBank accession number NM-002570) (SEQ ID NO: 40),
(D8) neuropeptide Y receptor Y1 (GenBank accession number NM-000909) (SEQ ID NO: 41) For all B1~B7 gene or EST described genes or all and claim 1 of the EST of A1~A15 according to claim 1, DNA containing all or part of the nucleotide sequence of that was spotted DNA Microarray. For all D1~D8 gene or EST described genes or all and claim 1 of the EST of C1~C11 according to claim 1, DNA microarrays DNA comprising all or part of the nucleotide sequence was spotted .   A method for evaluating the effect of tamoxifen using the DNA microarray according to any one of claims 1 to 3.   The method to evaluate the estrogen activity inhibitory effect of tamoxifen using the DNA microarray of any one of Claim 1 to 3.   A method for screening a substance having an action of inhibiting the gene or EST on the DNA microarray from responding to estrogen in the same manner as tamoxifen, using the DNA microarray according to any one of claims 1 to 3.   A method for screening a substance having an effect of inducing the gene or EST on the DNA microarray to respond to estrogen in the same manner as tamoxifen, using the DNA microarray according to any one of claims 1 to 3.   A method for screening for a substance having an action of inducing or suppressing that a gene or EST on a DNA microarray responds to estrogen, similar to tamoxifen, using the DNA microarray according to any one of claims 1 to 3. .   A method for screening a substance that suppresses the expression of a gene or EST on a DNA microarray in the same manner as tamoxifen, using the DNA microarray according to any one of claims 1 to 3.   A method for screening a substance that induces the expression of a gene or EST on a DNA microarray in the same manner as tamoxifen, using the DNA microarray according to any one of claims 1 to 3.   A method for screening for a substance that acts in the same manner as tamoxifen on the expression of a gene or EST on the DNA microarray using the DNA microarray according to any one of claims 1 to 3.   The expression of the gene according to any one of claims 4 to 11, comprising analyzing the expression of the gene on the DNA microarray according to any one of claims 1 to 3 by real-time PCR targeting the EST. the method of.