CN1696154A - Breast cancer related protein, gene encoding the same, and method of diagnosing breast cancer using the protein and gene - Google Patents

Abstract

An isolated protein having the amino acid sequence of SEQ ID No. 4 and having apoptosis-inducing activity and its encoding gene are provided. Also, a microarray having a substrate immobilized with genes or fragments thereof is provided. Also, a method for diagnosing breast cancer using an antibody specifically binding to the protein and a method for diagnosing breast cancer by determining whether the gene is expressed in cells are provided.

Description

Breast cancer-associated protein, gene encoding the same, and method for diagnosing breast cancer

Background technique

1. Field of invention

The present invention relates to a breast cancer related protein (BCRP) with the activity of inducing apoptosis, its coding gene, and a microarray with fixed BCRP gene fragments. Also, the present invention relates to a method for diagnosing breast cancer by using an antibody specifically recognizing BCRP and a method for diagnosing breast cancer by determining whether BCRP gene is expressed.

2. Description of related fields

When considering advanced cancers, breast cancer is second only to lung cancer in being the most commonly diagnosed and occurring in women. The incidence of breast cancer has increased steadily over the past 50 years, especially in South Korea. Several risk factors increase a woman's chances of developing breast cancer. These factors include aging, previous history of breast cancer, exposure to radiation, family history of breast cancer, social and economic class, pregnancy, menarche, menopause, or first pregnancy after age 30.

While breast cancer is known to be a heterogeneous disease and estrogen induces different breast tumors, there are many other recognized and unknown factors. Identified changes in oncogenes include amplification of the HER-2 and epidermal growth factor receptor genes and overexpression of cyclin D1. Overexpression of oncogenes is associated with a rather mild prognosis. Similarly, genetic alterations or loss of tumor suppressor genes, such as p53, have been shown to be associated with breast cancer and are associated with a milder prognosis. Researchers have identified two genes, called BRCA1 and BRCA2, as predictors of familial breast cancer in premenopause. Early diagnosis of breast cancer is important to ensure the best possible treatment outcome. Many countries with advanced healthcare systems have breast cancer screening programs. Information related to treatment selection and prognosis, eg, measurement of estrogen and progesterone receptor status, is also included in this procedure.

The main goals in the treatment of breast cancer are to improve the success rate of early detection, to discover novel non-invasive markers that can track disease progression and identify recurrence, and to discover improved treatments for progressive disease that still has a very low 5-year survival rate. There is a need to identify more specific targets of cancer cells, ideally those expressed on the surface of tumor cells, to attack tumor cells with new and promising approaches such as immunotherapy and directed toxins.

The present invention was accomplished by discovering a protein specifically expressed in breast cancer while studying breast cancer-specific single nucleotide polymorphisms (SNPs).

Summary of the invention

The present invention provides isolated proteins that are specifically expressed in breast cancer cells.

The present invention also provides the nucleic acid sequence encoding the protein, and the microarray immobilized with the nucleic acid sequence encoding the protein or fragments thereof.

The present invention also provides a method for diagnosing breast cancer by using an antibody specifically binding to the protein.

The present invention also provides a method for diagnosing breast cancer by determining whether the gene encoding the protein is expressed in breast tissue.

According to one aspect of the present invention, there is provided an isolated protein having the amino acid sequence of SEQ ID No.4 and specifically expressed on breast cancer.

According to another aspect of the present invention, there is provided a method for detecting the presence of breast cancer, the method comprising: reacting an anti-BCRP antibody specifically binding to the protein of the present invention with a polypeptide sample isolated from human breast tissue.

According to another aspect of the present invention, there is provided a polynucleotide encoding the protein of the present invention.

According to another aspect of the present invention, there is provided a polynucleotide or its complementary polynucleotide for the diagnosis or treatment of breast cancer, comprising at least 10 consecutive nucleotides derived from the polynucleotide of the present invention, immobilized with the polynucleotide Microarrays of polynucleotides or their complementary polynucleotides, and kits comprising the polynucleotides or their complementary polynucleotides.

According to another aspect of the present invention, a method for diagnosing breast cancer is provided, the method comprising: obtaining a breast tissue sample from the sample; measuring the expression level of the protein of the present invention in the breast tissue sample and judging the presence of breast cancer according to the result.

Brief description of the drawings

The above and other features and advantages of the present invention will become more apparent by describing a detailed exemplary embodiment thereof with reference to the accompanying drawings:

Figure 1 shows the Northern blot results of different normal cells with breast cancer related protein (breast cancer related protein) (BCRP) gene-specific probe;

Figure 2 shows the results of Northern blotting of different cancer cells with BCRP gene-specific probes;

Figures 3 to 5 show the sites of BCRP expression in cells, as observed in each colon cancer cell line clone A (CA), primary cultured normal kidney cells and HEK 293 cell line transfected with BCRP-pFLAG vector DNA FITC fluorescence for identification;

Figure 6 shows the expression levels of BCRP in CA cell lines transfected with the BCRP gene and the expression levels of different apoptosis-related genes when BCRP is overexpressed, identified by reverse transcription polymerase chain reaction (RT-PCR);

Figure 7 shows the effect of overexpression of the BCRP gene in HEK 293, CA and CX-1 (referred to as A, B and C, respectively) cell lines on the proliferation of the cell lines, as identified by a cell proliferation assay (MTT assay);

Figures 8 to 10 show the results of FACS analysis of HEK 293, CA and CX-1 transfected with BCRP-pFLAG vector DNA, which determined the effect of overexpression of transfected BCRP on cell lines by performing apoptosis assays;

Figure 11 and Figure 12 show the expression level of BCRP, which is identified by RT-PCR by isolating RNA from breast cancer tissues and normal tissues of two patients, and using the oligonucleotides of SEQ ID Nos.4 and 5 as primers;

Figure 13 is the result of the Northem blot assay of BCRP, using total RNA from breast tumor tissues of three patients to analyze the expression level of BCRP;

Figure 14 shows that when the anticancer agent paclitaxel (Taxol) is used to treat the MDA-MB-231 cell line to induce apoptosis, the change of the cell form; and

Figure 15 shows the effect of paclitaxel on BCRP gene expression as identified by RT-PCR using RNA extracted from the cells in Figure 14 .

Detailed description of the invention

The isolated protein according to one embodiment of the present invention has the amino acid sequence of SEQ ID No. 4 and has the activity of inducing apoptosis specifically expressed in breast cancer. The protein according to one embodiment of the present invention (hereinafter, also referred to as breast cancer-related protein (BCRP)) is a membrane protein specifically expressed in heart tissue of normal tissue. In addition, this BCRP is specifically expressed in breast cancer tissues in various cancer tissues. Therefore, by determining whether BCRP is expressed, breast cancer can be detected.

When BCRP is overexpressed in cells, it enhances the expression of at least one of p53 and p21.

The method for detecting breast cancer according to another embodiment of the present invention comprises reacting an anti-BCRP antibody specifically binding to BCRP with a polypeptide sample derived from human breast tissue. Breast cancer is judged when the expression level of BCRP is higher than that in normal tissues, preferably higher than 3%, 5%, 10% or higher than 15%. Methods for producing antibodies against specific protein antigens are well known in the art, and anti-BCRP antibodies in the present invention can also be produced by conventional methods.

The polynucleotide according to another embodiment of the present invention encodes an isolated protein having the amino acid sequence of SEQ ID No. 4 and having the activity of inducing apoptosis. The polynucleotide can have the nucleotide sequence of SEQ ID No.3. The polynucleotide is used for expressing BCRP and judging the existence of breast cancer by studying whether the BCRP is expressed.

According to another embodiment of the present invention, the polynucleotide or its complementary polynucleotide for the diagnosis or treatment of breast cancer comprises at least 10 consecutive polynucleotides derived from the nucleotide sequence of SEQ ID No.3 Nucleotides. A polynucleotide may be 10-100, preferably 10-50 nucleotides in length. Such polynucleotides can be used as primers or probes.

A microarray for diagnosing breast cancer according to another embodiment of the present invention has a substrate immobilized with a polynucleotide or its complementary polynucleotide for the diagnosis or treatment of breast cancer, and the polynucleotide or its complementary polynucleotide The nucleotides comprise at least 10 contiguous nucleotides derived from the polynucleotide having the nucleotide sequence of SEQ ID No. 3. The length of the polynucleotide may be 10-100, preferably 10-50 nucleotides, but is not limited thereto.

The kit for the diagnosis or treatment of breast cancer according to another embodiment of the present invention comprises at least 10 consecutive nucleotides derived from the polynucleotide having the nucleotide sequence of SEQ ID No.3.

A method for diagnosing breast cancer according to another embodiment of the present invention, comprising obtaining a nucleic acid sample from the breast tissue of the sample, and measuring the expression level of the protein with amino acids of SEQ ID No.4 in the breast tissue sample, and then determining the breast cancer according to the result the presence of cancer.

In this method, the expression level of the protein can be determined by various methods known in the art. In this method, whether the BCRP gene is expressed can be determined at the mRNA level by Northern blotting using a BCRP gene-specific probe. Alternatively, whether BCRP gene is expressed can be determined by extracting total RNA including mRNA, performing RT-PCR with BCRP gene-specific primers, and identifying the obtained product. However, the method of determining whether breast cancer is expressed is not limited thereto. Nucleic acid samples derived from breast tissue do not have to be purely purified nucleic acid samples, and in any method of analysis only the presence of nucleic acids useful for the analysis is required. For example, when PCR is used to identify the expression of the BCRP gene, a sample with lysed cells, which has not been subjected to nucleic acid isolation, can be used.

When the expression level of BCRP is higher than that in normal breast tissue cells, it can be judged as breast cancer.

Nucleic acids or proteins specifically expressed in breast cancer cells were searched for different nucleotide sequences selected from commercially available nucleic acid or protein databases. As a result, the nucleic acid or protein speculated to be specifically expressed only in breast cancer cells was identified, and the BCRP nucleic acid or protein was screened. Next, the BCRP full-length gene was identified by searching the cDNA library and its nucleotide sequence was analyzed. The specific expression of this gene in the patient's breast cancer cell line and breast cancer cells was identified by Northern blot assay (Figure 11 and Figure 12). It was also identified by repeated assays that the expression level in breast cancer cells was twice as high as in normal cells (Figure 12). The location of expression of the protein encoded by the BCRP gene in the cells was identified by immunocytochemistry. In one method, the gene of the protein is recombined with the pFLAG vector, and the animal cell line is transfected with the obtained recombinant vector, and then, the expression is identified by the pFLAG fluorescence system (Fig. 3, 4 and 5). Furthermore, BCRP was overexpressed in cells, and the effect of BCRP on cells was studied to identify the function of BCRP. To this end, animal cell lines were transfected with the gene encoding BCRP, from which total RNA including mRNA was extracted and used as template for RT-PCR. The expression levels of BCRP gene and other genes related to apoptosis such as p53 and p21 were monitored by RT-PCR (Fig. 6). The promoter structure of the BCRP gene was also identified.

The present invention will be described in more detail with reference to the following examples. The following examples are for illustrative purposes and are not intended to limit the scope of the invention.

Example

Example 1: Searching for BCRP genes based on SNP data

1. Search for BCRP gene

The nucleotide sequence of the site where the gene related to breast cancer is located on the SNP is found by separate research, searched by database (NCBI) search and analysis, and thereby obtains information that can design primers for amplifying the gene.

2. Amplification of BCRP gene fragment

Primers were designed using the obtained base sequence information (SEQ ID Nos. 1 and 2) to amplify DNA surrounding the searched SNP.

Then, PCR was performed using genomic DNA as a template and the synthesized primer set as primers to amplify a 239bp BCRP gene fragment. In PCR, use 10 pmol each of forward primer and reverse primer and 200 pg-1 μg of genomic DNA template, and then repeat the reaction 35 times at 95°C for 40 seconds, at 57°C for 40 seconds, and at 72°C for 1 minute. Results were confirmed via 1% agarose gel electrophoresis and amplification of the expected DNA fragment of 239 bp was identified. The amplified DNA fragment was used as a probe to search for the full-length BCRP gene.

3. Search the full-length gene of BCRP via cDNA library

A human fetal brain cDNA library (λtriplrEx library, available from Clontech Corporation) was used as the cDNA library, and the study procedure followed the manufacturer's (PT3003-1) protocol. A brief research procedure is as follows.

PCR was performed to obtain mRNA, while cDNA library searches were performed using previously obtained PCR products as probes. The cells used for the search were E. coli XL-1 blue cells commonly used in the art.

First, in the titer determination of the library, both groups were set at 2.0×10 ⁹ pfu/ml. Then, spread the cDNA library on the E. coli XL-1 blue plate. Typically, 2-5 x 10 ⁴ pfu/150 mm are plated for cDNA libraries. Then, the lambda phage (phage) was transferred to a positively charged nylon membrane. Filter hybridization was performed using DNA-labeled probes with random primers labeled with radioisotope-labeled dCTP ([α- ³² P]dCTP, 3000 Ci/mmol), and hybridization results were measured by detecting signals of the labeled probes. As a result, positive clones were obtained.

4. Analysis of BCRP full-length gene base sequence and protein sequence prediction

By analyzing with an automatic sequence analyzer (ABI 3700), the base sequence of the BCRP full-length gene obtained from the above clone was identified. Furthermore, the deduced protein sequence of the gene was determined using NCBI and the new GENSCANW net program. The nucleotide sequence of the BCRP gene is identical to SEQ ID No.3, therefore, the amino acid sequence of the encoded protein is identical to SEQ ID No.4. The SNPs used to search for BCRP are located in the promoter region, and the BCRP genomic gene is located between 16032 and 96546 and consists of 3 introns and 4 exons.

Example 2: Identification of BCRP Gene Expression in Cells and Tissues

1. Identification of BCRP gene expression in cells by Northern blotting

Using the PCR product obtained in Example 1 as a probe, a number of human normal tissues and tumor tissues (available from Clontech Corporation) were subjected to Northern blot analysis. The results are shown in Figures 1 and 2. Referring to Figures 1 and 2, the BCRP gene is specifically expressed only in heart tissue in normal tissue and in breast cancer tissue in tumor tissue. Therefore, it is evident that the study of BCRP gene expression can be used to detect the presence of breast cancer. In Fig. 1, the tissues used in Northern blotting are brain heart, heart, skeletal muscle, colon, thymus, spleen, kidney, liver, small intestine, placenta, lung and peripheral blood leukocytes. In Fig. 2, the cancer tissues used in the Northern blotting method were cell lines of breast, ovary, uterus, lung, kidney, stomach, colon, and rectum (Northern blotting of various tissues manufactured by Clontech Corporation). 1.00kb and 1.2kb are at the top of Figure 1 and Figure 2, respectively, indicating marker size and blot results are shown at 1.37kb.

Specific manipulations for Northern blotting were performed as follows.

(1) Preparation of radiolabeled probes

Probes were prepared using DNA labeling with random primers (Roche Random Primer DNA Labeling Kit, #1004760). About 25 ng of purified BCRP PCR product and isotope [α- ^32P ]dCTP, 250 µCi (available from BMS Corporation) were used.

(2) Pre-hybridization with a hybridization bottle

Nylon membranes were prehybridized for 30 minutes in 7 ml of ExpressHyb solution (#8015-1, available from BDClontech) preheated to 68°C.

(3) Denaturation of radiolabeled probes

Radiolabeled probes were heated at 95-100°C for 8-10 minutes and then quickly placed on ice.

(4) hybridization

100ml of fresh ExpressHyb solution was mixed with the radiolabeled probe. The prehybridization solution was removed from the hybridization vial containing the nylon membrane, and fresh ExpressHyb solution mixed with the radiolabeled probe was added. Then, shake flasks were incubated at 68°C for 1 hour.

(5) washing

Wash the nylon membrane in the bottle with washing solution 1 at room temperature for 30-40 minutes, and then wash with washing solution 2 at 50°C for 40 minutes. Then, remove the nylon membrane from the bottle and dry to the point where it retains a little moisture, then wrap it in plastic.

(6) The nylon film was placed in X-ray film and exposed to -70°C. After 1-2 days, the nylon membrane was removed and the bands were identified.

2. Identification of BCRP Gene Expression in Tissues

The expression of the BCRP gene was identified in the lesioned and non-lesional fractions of breast cancer patients.

Figures 11 and 12 show the results of RT-PCR with RNA isolated from cells obtained from breast cancer tissues of two patients, and using oligonucleotides of SEQ ID Nos. 4 and 5 as primers. Referring to Figures 11 and 12, the expression of BCRP gene was increased in breast cancer tissue compared with that in normal tissue, while the expression of p53 was significantly increased, which was found to be associated with breast cancer and was selected as a comparative gene.

Figure 13 shows the results of Northern blotting of RNA from breast tumor tissues and normal tissues. Referring to Fig. 13, the expression of BCRP gene was enhanced (about 1.8 kb). In Fig. 13, lanes 1 to 3 are the results of breast tumor tissues from different donors, and lane 4 is the result of normal breast tissues. As a result of comparing the average expression level of lanes 1 to 3 with the expression level in normal breast tissue, it can be seen that the expression level of BCRP gene in breast cancer tissue is about 2.05 times higher than that in normal tissue.

Embodiment 3: Identification of the expression position of BCRP gene in the cell

The expression position of BCRP gene in cells was identified by immunocytochemical method. For this, the BCRP gene was first cloned into the pFLAG vector (Sigma, Amherst, NY). The cloning steps are as follows. The BCRP gene of SEQ ID No. 3 was digested with Not I enzyme and Sal I enzyme, and the pFLAG vector was digested with the same enzymes as above, and then the digested BCRP gene and pFLAG vector were ligated. Each cell line was transfected with cloned BCRP-pFLAG vector DNA using Lipofectin 2000. The transfected cells were cultured for 48 hours at 37°C in 5% CO ₂ to enable expression of the BCRP gene. Then, the cultured cells were fixed on the plate with 3.5% paraformaldehyde. For intracellular staining, cells were permeabilized by 0.1% Triton X-100. Block blank areas with 1% BSA blocking solution. The Flag-specific antibody was reacted with the plate on which the cells were immobilized, so that the flag-specific antibody (anti-FLAG M2) specifically bound to BCRP-Flag. Finally, a FITC-conjugated secondary antibody (anti-mouse IgG-FITC) was reacted with the BCRP-Flag-primary antibody conjugate. The location of BCRP expression was identified by fluorescence generated by FITC using fluorescence microscopy. As a control, a non-transformed cell line was tested using a Flag-specific antibody in the same manner as above.

The results are shown in Figures 3 to 5. Figures 3 to 5 show the observed FITC fluorescence of colon cancer cell line Clone A (CA), primary cultured normal kidney cells and HEK293 cell line transfected with BCRP-pFLAG vector DNA. As shown in Figures 3 to 5, BCRP was observed in the cell membranes of all three cell lines.

Therefore, it was judged that BCRP according to the embodiment of the present invention was specifically expressed in the cell membrane.

Example 4: Effect of BCRP Gene Expression on Gene Expression in Cell Lines

The effect of the expression of the BCRP gene on the expression of the gene in the cell lines was identified. To this end, the presence of BCRP gene mRNA was first identified by a Northem blot test. Then, the effect of the expression of the BCRP gene on the expression in cells of genes known to be associated with cancer and apoptosis was studied in actual cell lines. First, the CA (Colon A) cell line was transfected with the BCRP-pFLAG carrier DNA prepared as above using Lipofectin2000, and the transfected cell line was cultured at 37° C. in 5% CO ₂ for 48 hours. Then, total RNA including mRNA was extracted and subjected to RT-PCR. β-actin was used as a control. Primer sets capable of amplifying BCRP, β-actin, p53, p21, CytC, caspase 5, caspase 3, and Apaf 1 genes, respectively, were used as primers (Table 1). The expression level of each gene was identified by monitoring the amount of PCR product obtained from RT-PCR.

Table 1

Primer sequences used in the amplification of each gene Gene Primer sequence BCRP F: SEQ ID No.5 R: SEQ ID No. 6 β-actin F: SEQ ID No. 7 R: SEQ ID No. 8 P53 F: SEQ ID No.9 R: SEQ ID No. 10 P21 F: SEQ ID No. 11 R: SEQ ID No. 12 CytC F: SEQ ID No. 13 R: SEQ ID No. 14 Caspase 5 F: SEQ ID No. 15 R: SEQ ID No. 16 Caspase 3 F: SEQ ID No. 17 R: SEQ ID No. 18 Apaf1 F: SEQ ID No. 19 R: SEQ ID No. 20

RT-PCR was performed as follows: total RNA including mRNA extracted from a cell line overexpressing the BCRP gene was reverse-transcribed using Superecript II reverse transcriptase (available from Invitrogen), thereby obtaining cDNA. 5 ng of the obtained cDNA was subjected to PCR using Taq polymerase as a template and using 10 pmol each of a forward primer and a reverse primer (refer to Table 1). In PCR, the reaction was repeated 30 times at 95°C for 40 seconds, at different annealing temperatures for 40 seconds and at 72°C for 1 minute according to the gene to be amplified. Suitable annealing temperatures are 58°C for β-actin, caspase 5 and BCRP, and 52°C for p53, p21, CytC, caspase 3 and Apaf 1. Results were identified by 1% agarose gel electrophoresis and are shown in FIG. 6 . Referring to Fig. 6, the expression of p53 was increased, as was the expression of p21, which is known as a transient mediator of p53-dependent growth inhibition. However, the expression of other genes did not show specific changes.

Example 5: Effect of Overexpression of BCRP Gene on Cell Line Expansion and Apoptosis

1. MTT test

The effect of overexpression of BCRP gene in cells on cell line expansion and apoptosis was identified by MTT assay. The MTT assay is a method of measuring the absorbance of formazan produced by reducing MTT with mitochondrial dehydrogenase in living cells. The measured absorbance reflects the concentration of metabolically active cells. The cell lines used in this assay were Clone A and CX-1 (colon cancer cell lines), and the normal kidney cell line, HEK293. BCRP-pFLAG vector DNA was transfected with Lipofectin2000 as in Example 4. Controls used in this assay were a vector control transfected with pFLAG only, a nothing control that was not transfected but treated the same, and a blank control that had no cells.

Results were obtained from two replicate experiments. Less cell expansion occurred in the CA and CX-1 cell lines than in the controls. In other words, cell growth was inhibited in CA and CX-1 cell lines compared to controls. Likewise, cell expansion occurred less in the HEX 293 cell line than in the colon cancer cell lines (CA and CX-1 ) compared to the other two controls. The results are shown in Figure 7, which shows the results of the evaluation of the effect of overexpression of BCRP on cell line expansion in HEK 293, CA and CX-1 (represented by A, B and C, respectively) cell lines.

2. Cell Apoptosis Assay

HEK 293, CA and CX-1 cell lines were used to study the effect of BCRP gene expression on apoptosis by flow cytometry. A vector control transfected with only the pfFLAG vector was used as a control.

As a result of two repeated experiments, both CA and CX-1 showed less apoptosis than the control in both experiments, while HEK 293 showed weak or no apoptosis. Figures 8 to 10 show the test results. Figure 8 to Figure 10 show the FACS analysis results of HEK 293, CA and CX-1, respectively.

Example 6: Effect of apoptosis on BCRP gene expression in human breast cancer cells

It is conventionally known that when human breast cancer cells are treated with the anticancer agent paclitaxel, apoptosis is induced. In this example, to investigate the expression level of BCRP gene when apoptosis was induced, the MDA-MB-231 cell line was treated with paclitaxel to induce apoptosis, and RT-PCR was performed in the same manner as above.

Figure 14 shows the changes in cell form when the MDA-MB-231 cell line was treated with paclitaxel. It can be seen from B of Fig. 14 that paclitaxel induces apoptosis.

Figure 15 shows the identification of the effect of paclitaxel on BCRP gene expression by RT-PCR. Referring to Figure 15, paclitaxel enhanced the expression of BCRP gene.

It is also apparent from the above results that the BCRP gene according to one embodiment of the present invention suppresses cell proliferation. Furthermore, although BCRP did not strongly induce apoptosis, it was slightly induced compared to the control. In addition, considering the results of BCRP expression in paclitaxel-treated cell lines as indirect evidence, it was postulated that BCRP was associated with apoptosis. Also, it was confirmed in Example 4 that the BCRP gene enhances the expression of p53 and p21. Therefore, it was judged that the overexpression of BCRP gene enhanced the expression level of p53, and the activation of p53 enhanced the expression level of p21.

The isolated protein and the nucleic acid encoding it according to the embodiments of the present invention can be used for diagnosing breast cancer and developing drugs targeting the protein.

The method of diagnosing breast cancer, using an antibody specifically binding to BCRP according to one embodiment of the present invention, can be used to effectively diagnose breast cancer.

The method of diagnosing breast cancer can be used to effectively diagnose breast cancer by measuring the expression level of BCRP gene according to one embodiment of the present invention in cells.

Microarrays according to one embodiment of the present invention can be used in various assay methods such as assays to detect the presence of breast cancer.

While the invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made without departing from the spirit of the invention as defined in the following claims: spirit and scope.

Sequence Listing

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<400>19

gggtttcagt tgggaaacaa 20

<210>20

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> Reverse primer for Apaf 1 amplification

<400>20

cacccaagag tcccaaacat 20

Claims (14)

1. an isolated protein, which has the amino acid sequence of SEQ D No.4 and is expressed specifically to breast cancer. 2. The isolated protein of claim 1 which is associated with apoptosis. 3. The isolated protein of claim 1, which enhances expression of at least one of p53 and p21 in a cell. 4. A method for detecting the presence of breast cancer, the method comprising reacting an anti-BCRP antibody specifically binding to the protein of claim 1 with a polypeptide sample derived from human breast tissue. 5. A polynucleotide encoding the protein of claim 1. 6. the polynucleotide of claim 5, it has the nucleotide sequence of SEQ ID No.3. 7. A polynucleotide or its complementary polynucleotide for the diagnosis or treatment of breast cancer, comprising at least 10 consecutive nucleotides, which are derived from a polynucleotide having the nucleotide sequence of SEQ ID No.3 glycosides. 8. The polynucleotide of claim 7, which is 10-100 nucleotides in length. 9. The polynucleotide of claim 8 which is a primer or a probe. 10. A microarray for diagnosing breast cancer, having a substrate immobilized with the polynucleotide of claim 8 or a complementary polynucleotide thereof. 11. A kit for diagnosing breast cancer comprising the polynucleotide of claim 8 or a complementary polynucleotide thereof. 12. A method for diagnosing breast cancer, the method comprising Obtain breast tissue samples from samples and Determining the expression level of the protein of claim 1 in a breast tissue sample and judging the presence of breast cancer based on the result. 13. The method of claim 12, wherein the expression level of the protein is determined by Northern blotting or electrophoresis. 14. The method of claim 12, wherein breast cancer is determined when the expression level of said protein is higher than that in normal breast tissue.

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