KR20130045888A - Composition for diagnosing and /or treating cancer and the kit - Google Patents

Abstract

PURPOSE: A composition and a kit for diagnosing and/or treating cancer are provided to reduce telomerase activity which is enhanced by HCCR-1 in RKO cells. CONSTITUTION: A composition for treating colon cancer contains an amino acid sequence of sequence number 3 as an active ingredient. A composition for diagnosing cancer contains a protein containing an amino acid of sequence number 1 or 2 as a marker for diagnosing cancer. The composition contains a monoclonal antibody which binds to an amino acid residue of sequence number 1 or 2. A kit for diagnosing cancer contains the protein. A kit for diagnosing cancer contains the monoclonal antibody.

Description

Composition and kit for diagnosis and/or treatment of cancer {COMPOSITION FOR DIAGNOSING AND /OR TREATING CANCER AND THE KIT}

The present invention relates to a composition for diagnosis and/or treatment of cancer and a kit thereof.

The product of the tumor suppressor gene inhibits transformation from normal cells to cancer cells, and when this function is lost, the cell leads to malignant transformation (Klein, G., FASEB J. , 7 , 821- 825 (1993)). In order for cancer cells to form cancer, the normal copy number function of the cancer suppressor gene must be lost. Modifications in the coding sequence of the p53 cancer suppressor gene were found to be the most common genetic changes in human cancer (Bishop, JM, Cell , 64 , 235-248 (1991); and Weinberg, RA, Science , 254 , 1138-1146 ( 1991)). However, the p53 mutation reported in cervical cancer is only in the range of 2% to 11% (Crook, T. et al., Lancet , 339 , 1070-1073 (1992); And Busby-Earle, RMC et al., Br, Br. j. Cancer , 69 , 732-737 (1994)), only a portion of cervical cancer tissues are thought to exhibit this p53 mutation. Therefore, the present inventors use the mRNA differential display (DD) method, which more efficiently expresses genes differentially expressed between normal cervical tissue and cervical cancer, to create new cancer suppressor genes in normal cervical tissue. It was studied carefully to separate.

Oncogene It is known that HCCR-1 acts as a negative regulator of p53 and acts on tumorigenesis in various organs, including the large intestine (Ko, J. et al . Oncogene 22, 4679-4689 (2003); Ko, J. et al . . Oncogene 23, 1950-1953 (2004); Yoon, SK et al . Cancer Res . 64, 5434-5441 (2004)) To understand the tumorigenic pathway of HCCR-1, the present inventors performed a yeast two-hybrid screen, and HCCR-1 is the DP1 (Deleted in polyposis 1) gene. Revealed that it interacts with. The HCCR-1 interacting domain in DP1 is 61-120 residues. These two proteins coexist in mitochondria, but the expression of HCCR-1 shows a negative correlation with the expression of DP1 in colon cancer. The present inventors investigated the usefulness of serum HCCR-1 in colon cancer patients using a sandwich ELISA method. The sensitivity of HCCR-1 to detect colon cancer (76.0%) is much greater than that of CA19-9 (32.0%), and is particularly effective in the early stages of colon cancer. Loss of heterozygosity in the DP1 gene is often observed in colorectal cancer (57.1%). Studies show that DP1 plays a role as a tumor suppressor in colon tumorigenesis; That is, DP1-transfected RKO cells showed growth inhibition, epitosis, reduced telomerase activity, and up-regulation of p53, p21, MDM2 and Bax. This phenomenon was reversed when HCCR-1 was overexpressed. In addition, the present invention describes that DP1 plays a role of a tumor suppressor, particularly in colorectal cancer, and that overexpressed HCCR-1 inhibits the tumor suppressor action of DP1 by interaction.

Mutation of cancer genes or tumor suppressor genes is one of the main mechanisms of tumor formation. Therefore, revealing new tumor-associated genes and their mechanisms of action is important in overcoming malignant tumor diseases. Recently, the present inventors discovered a new oncogene HCCR, which is HCCR-1 (GenBank accession number AF 195651) and HCCR-2 It is classified into two types (GenBank accession number AF 315598) (Ko, J.et al.Oncogene 22, 4679-4689 (2003)). Cells overexpressing HCCR-1 or HCCR-2 formed tumors in nude mice, and HCCR transgenic mice developed breast cancer and metastasis (Ko, J.et al.Oncogene 22, 4679-4689 (2003); Ko, J.et al . .Oncogene 23, 1950-1953 (2004)). In addition, HCCR was found to negatively regulate the p53 tumor suppressor gene (Ko, J.et al.Oncogene 22, 4679-4689 (2003); Ko, J.et al . .Oncogene 23, 1950-1953 (2004)).

HCCR-1 is overexpressed in several types of human malignancies, including colorectal cancer (Ko, J. et al. al . Oncogene 22, 4679-4689 (2003)). In order to understand the HCCR-1 tumorigenic mechanism, the present inventors performed a yeast two-hybrid screen, and the DP1 protein encoded by the C5orf18 (GenBank accession number NM_005669) gene and interacting with HCCR-1 was identified. I sympathized. In particular, the DP1 gene is located near the adenomatous polyposis coli (APC) gene. APC One of the well-known tumor suppressor genes is mutated in the germ cell DNA of patients with familial adenomatous polyposis (FAP), and somatic mutations or allele deletions of APC are known in accidental colon cancers. In severe FAP, a region of about 100 kb-sized on the 5q22 chromosome was generally deleted. In addition to the APC gene, DP1 is located in this submicroscopic region of the defect, but a putative tumor suppressor gene mutated from another colon cancer (MCC) exists outside this common region of the defect. In contrast to the severe form of FAP, patients with mild FAP show APC and MCC mutations in this region with no chromosomal defects. The data led the inventors to study the possibility that HCCR-1 could contribute to colon tumor formation by negative regulation of DP1 through interaction.

It is an object of the present invention to provide a composition for diagnosis and/or treatment of cancer.

Another object of the present invention is to provide a kit for diagnosing cancer.

In order to achieve the above object, the present invention provides a composition for cancer diagnosis comprising a protein comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as a marker for cancer diagnosis.

In addition, the present invention provides a composition for diagnosis of cancer comprising a monoclonal antibody that binds to the amino acid residues of SEQ ID NO: 1 or SEQ ID NO: 2.

In addition, the present invention provides a cancer diagnostic kit comprising a protein having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as a marker for cancer diagnosis.

In addition, the present invention provides a kit for diagnosis of cancer comprising a monoclonal antibody that binds to the amino acid residues of SEQ ID NO: 1 or SEQ ID NO: 2.

In addition, the cancer that can be diagnosed in the present invention is preferably colon cancer, brain cancer, blood cancer, kidney cancer, or liver cancer, and colon cancer is most preferred.

In addition, the present invention provides a composition for treating cancer comprising the DP1 protein having the amino acid sequence of SEQ ID NO: 3.

Hereinafter, the present invention will be described.

In the present invention, the interaction of HCCR-1 and DP1 by the yeast two-hybrid screen was confirmed by glutathione-S-transferase (GST) pull-down experiments and immunoprecipitation studies in vitro. In order to investigate the domain of the interaction between DP1 and HCCR-1, the present inventors developed six GST fusion constructs of full-DP1p, DP1p1-165, DP1p1-60, DP1p61-185, DP1p61-165 and DP1p121-185. Was prepared. As a result, the DP1 binding site for HCCR-1 was depicted as amino acid residues 61-120 (FIG. 1A ). This region is included in the TB2-DP1-HVA22 domain of DP1, which may play an important role in this interaction. However, the biological significance of the TB2-DP1-HVA22 domain is still unclear. In order to confirm the interaction between HCCR-1 and DP1, the present inventors prepared a DP1-Myc fusion protein and cultured the protein extract and the fusion protein extracted from 293 cells transfected with HCCR-1 fused to V5 (Invitrogen). I did. The HCCR-1 protein is specifically coprecipitated with DP1 (Figure 1B). These results indicate that DP1 naturally interacts with the HCCR-1 oncogene.

The present inventors performed cell differentiation analysis and confirmed the mitochondrial location of HCCR-1 in HEK293 cells (Fig. 1c). Subcellular fractionation of RKO colorectal cancer cells co-expressed with HCCR-1 and DP1 indicates that these two proteins coexist only in mitochondria (Fig. 1D). CDNA constructs were prepared in which HCCR-1 and DP1 were tagged to V5 (top panel) and myc (bottom panel), respectively. The HCCR-1 protein stably expressed in RKO cells is only detected in the mitochondrial fraction (upper panel), and the DP1 protein is also mainly found in the mitochondrial fraction, while a small amount is found in the protoplasm and nucleus (lower panel). These results also explain their interactions and when interacting with DP1 the position of HCCR-1 does not change and similarly the position of DP1 does not change when the protein is co-expressed with the binding protein HCCR-1.

Northern blot analysis was performed to investigate the expression of HCCR-1 in colon tissues and cell lines. HCCR-1 (∼2.1 kb) and HCCR-2 (∼2.0 kb) mRNAs were overexpressed in primary colon cancer tissues compared to colon cancer cell lines (RKO and HT-29 cells) and their normal tissues (Fig. 2a). .

In order to confirm whether HCCR-1 expression is involved in human colon cancer, JE33 and I87, which are monoclonal antibodies to amino acids 167-360 of HCCR-1, were generated. Binding regions on HCCR-1 for JE33 and I87 mAb were determined through time-dependent trypsin fragmentation pattern analysis of HCCR-1 using mass spectrometry. As structurally hidden areas are protected from trypsin, trypsin treatment often produces partial cut sections at the beginning of the treatment. Similarly, protein complexes protect each other from trypsin cleavage in the contact area. Well protected areas are not cut or are cut slowly compared to exposed areas. Based on this concept, the pattern of truncated fragments over time can be used to determine areas that are inaccessible or hidden by structures or other interacting molecules. Trypsin treatment of maltose binding protein (MBP)-HCCR-1 alone (control) and complexed with mAbs exhibited a similar fragmentation pattern except for the late appearance rate of some fragments. Compared to the control, the slowly cleaved fragments may be part of the region interacting with the counterpart protein, so the two fragments considered to be slowly cleaved fragments, namely amino acid residues 169-187 and amino acid residues 291-310, are JE33 and I87, respectively It will be an epitope of HCCR-1 that interacts with mAbs. The two fragments correspond to the sequences HFWTPKQQTDFLDIYHAFR (SEQ ID NO: 1) and AKLGIGQLTAQEVKSACYLR (SEQ ID NO: 2).

Using JE33 mAb, immunostaining experiments were performed on colon cancer and normal colon tissues from 21 patients. As a result, HCCR-1 was overexpressed in 21 colon cancer tissues. The immune response is mainly observed in cancer cells in a dominant form (Fig. 2B; left panel). There is no expression of HCCR-1 in the normal colon tissue (Fig. 2B; right panel). A larger number of samples may need to be investigated, but these results indicate that increased expression of HCCR-1 is associated with the incidence of human colorectal cancer.

One of the first tumor-associated antigens (TAAs) known as markers for colorectal cancer and other gastrointestinal tumors is the glycoprotein calcinoembryonic antigen (CEA). CEA is also expressed in several extra-intestinal tumors such as lung, breast, ovarian and bladder cancer. Determining serum CEA levels for detection of cancers was thought to have excellent effects such as accuracy, regeneration and economy. This has led many to believe that CEA could be used as a serological screening method for early colorectal cancer (CRC) detection. However, subsequent studies did not have useful sensitivity or specificity for this ability (Pokorny, RM, Hunt, L. & Galandiuk, S. Dig. Surg. 17, 209-15 (2000); Crawford , N. P. ., Colliver , D. W. & Galandiuk , S. J. Surg . Oncol . 84, 239-48 (2003)). Especially in the stage I CRC, its sensitivity/specificity was very low. Tumor-associated glycoprotein-72 (TAG-72), a high molecular weight mucin-like glycoprotein expressed in several human cancers; Carbohydrate antigen 19.9, an oligosaccharide associated with Lewis A blood group substances; And the other three TAAs, such as lipid-related cyclic acid, were also used as CRC markers. Like CEA, these tumor markers showed low sensitivity and specificity in CRC and were found to be ineffective as screening and diagnostic methods (Pokorny, RM, Hunt, L. & Galandiuk, S. Dig. Surg. 17, 209-15 ( 2000); Crawford , N. P. , Colliver , D. W. & Galandiuk , S. J. Surg . Oncol . 84, 239-48 (2003)) Early detection of colon cancer and prediction of treatment results based on the above results. Clinically applicable biomarkers for biomarkers have not yet been developed at CRC.

To confirm the possibility that HCCR-1 could be used as a biomarker for colorectal cancer, serum HCCR-1 levels were investigated in colorectal cancer patients, and the relationship between tumor stage and clinical pathological characteristics such as CA19-9 was investigated. . In colon cancer, the serum level of HCCR-1 (mean ± S.D., 8.0 ± 4.5 µg/ml) was much higher than that of the normal control group (4.7 ± 0.7 µg/ml) (P <0.0001). Although the receptor activating characteristic (ROC) curve analysis showed a value of 5.77 μg/ml with sensitivity, specificity and overall accuracy considered to be the most efficient cut-off in 100 randomly selected subjects, the present inventors showed a more convenient cut-off from a clinical point of view. 6 μg/ml was selected (Fig. 2c). Using a cut-off value of 6 μg/ml, 38 of 50 patients (76.0%) were detected by HCCR-1, but only 16 by CA19-9 (cut-off, 37 U/ml). Only people (32.0%) were detected. These results were quite different (P <0.0001). The specificity was 100% for both markers. Of the 34 patients diagnosed as negative by CA19-9, 24 patients (70.6%) were also accurately matched by HCCR-1 (Table 1). The HCCR-1 levels according to each step and the diagnostic results compared to CA19-9 are summarized in Table 2. The mean values of HCCR-1 levels did not differ significantly at different stages. The sensitivity to CRC stage I by the HCCR-1 marker was significantly higher compared to that of CA19-9. The rate of positive response to HCCR-1 was over 68% in stage I, but the rate to CA19-9 was only 5%. All positive response rates for HCCR-1 were much higher than rates for CA19-9 in each stage except stage II (Table 2). In order to confirm the sensitivity of HCCR-1 in precancerou colon disease, the present inventors analyzed 4 adenomas with benign bowel disease and 20 patients with 16 colitis. The mean serum level of benign colon diseases was 3.46 ± 2.45 µg/ml, and all patients showed 5.35 µg/ml and less, except for one patient with colitis (6.23 µg/ml).

<Diagnostic results of HCCR-1 and CA19-9>

<Diagnostic results compared to HCCR-1 levels and CA19-9 at each stage>

The HCCR-1 assay may have an advantage over the CA19-9 assay in that its level increases with disease progression and it is more benign in patients with early colorectal cancer. These results indicate that HCCR-1 may be a useful biomarker for molecular staging and early diagnosis of CRC.

In contrast to the overexpression of HCCR-1, the expression of DP1 was significantly reduced in colon cancer compared to normal tissues (FIG. 2D). The DP1 gene has two transcripts, 3.0 kb and 1.0 kb, respectively, and is widely expressed in several normal tissues (Fig. 2e), but the expression of DP1 is generally down-regulated in several human cancer cell lines (Fig. 2f). . These data showed that the putative tumor suppressor, the DP1 gene, was down-regulated in several human cancer cell lines, including colorectal cancer. It suggests that DP1 may act as a basic tumor suppressor in multiple human organs.

The present inventors investigated the frequency of allelic deletions in the DP1 locus. For this investigation, the D5S346 marker located within the DP1 gene was investigated together with two other markers (D5S519, D5S409) located near and far from the DP1 gene. LOH was often detected at locations around all three DP1s. Examples of LOH of the three microsetlite markers in colorectal cancer were described (FIG. 3A). In general, 66.6% (14 of 21) of 21 primary colorectal cancers showed LOH around the DP1 gene (FIG. 3B ). The LOH of the correct DP1 locus (D5S346) was also very high (57.1%).

In order to confirm the growth inhibitory function of DP1, DP1 was transfected into RKO colon cancer cells. DP1 transfection induced about 32% growth inhibition for 7 days in wild type or vector transfected RKO cells (FIG. 3C ). On the other hand, the rate of growth of HCCR-1-transfected RKO cells increased by about 88% for 7 days compared to cells transfected with only the vector (Fig. 3D). However, DP1-transfection-induced growth inhibition of HCCR-1-transfected RKO cell growth was about 63% for 7 days (Fig. 3D).

This inhibitory effect of DP1 on the growth of colorectal cancer cells was associated with an apoptosis process involving DNA fragmentation. Segmentation of DNA in the form of an oligonucleotide ladder was observed in DP1-transfected RKO cells in a time-dependent manner compared to cells transfected with wild-type or vector only (Fig. 3e). No DNA fragmentation was observed in HCCR-1-transfected RKO cells (Fig. 3e).

Epoptosis formation was also detected by annexin V/propidium-iodine (PI) (FIG. 3F). Annexin V/propidium-iodine (PI) flow cytometry results correlated with growth curve analysis and DNA fragmentation. The double count density dot plot of the annexin V/propidium-iodine (PI) assay shows the sensitivity of RKO cells to DP1 transfection. Compared to 3.8% of the vector transfected RKO cells, about 17.6% of the total DP1-transfected cells were in the initial process of epitosis (FIG. 3F). DP1 transfection also induced a late epoptosis process compared to vector only or HCCR-1 transfected RKO cells (FIG. 3F ). The annexin V-FITC/PI flow cytometry results of the present invention obtained a greater apoptosis rate by simultaneous annexin V/PI labeling in RKO cells for DP1 transfection. This result implies that DP1 induced epitosis-related plasma membrane lipids were changed by Annexin V.

It has been shown that many chemotherapeutic agents and ionizing radiation induce epoptosis, resulting in anticancer cytotoxicity (Thompson, CB Science 267, 1456-1462 (1995)). Exposure of mammalian cells to UVC can repair DNA or epoptosis. To neutralize the harmful effects of DNA damage. In order to measure short-wavelength light (UVC)-triggered apoptosis in DP1 transfected RKO cells, the cells were ^{treated with UVC (0, 50 and 100 J/m 2} ), respectively. In the FACS analysis of DP1 transfected RKO cells, the apoptosis sub G ₀ /G ₁ -fraction of the cell cycle is largely concentrated (Fig. 3G). FACS analysis of DP1 transfected RKO cells showed that the cell cycle epitosis sub G ₀ /G ₁ -the number of cells in the fraction was 0, 50 and 100 J/m ² After UVC treatment, wild-type or vector-transfected RKO cells Compared with, it increased by about 17, 32 and 60%, respectively. However, in HCCR- 1 -transfected RKO cells after 0, 50 and 100 J/m ² UVC treatment, about 3, 19 and 30% of cells, respectively, remained in the epitosis sub G ₀ /G ₁ -fraction. This flow cytometric result indicates that UVC exposure dose-dependently increases the amount of epitosis in DP1 transfected RKO cells when compared to wild-type or vector-transfected RKO cells. These results indicate that DP1 induced RKO cells are more sensitive to UVC light-triggered epitosis. Conversely, HCCR-1 induced cells are more resistant to UVC.

Down-regulation of LOH, DP1 expression, which occurs frequently in CRC, and a growth inhibitory effect or UVC sensitivity in DP1 transfected cells indicate that DP1 has a tumor suppressor effect in CRC.

Changes in the coding sequence of the *p53 tumor suppressor gene are the most common genetic changes in human cancers. Therefore, the present inventors investigated whether p53 expression is affected by DP1 and expression. In the present invention, DP1 transfection increased p53 RNA levels in wild-type RKO cells as well as in HCCR-1 co-transfected RKO cells (FIG. 4A). Unlike HCCR-1 transfected cells, p21 , MDM2 and Bax genes were P53 dependent up-regulated in DP1 transfected cells. In cells transfected with DP1 and HCCR-1 co-transfected, the HCCR-1 effect is compromised by DP1. Immunoprecipitation analysis indicates that p53 interacts with both HCCR-1 (Figure 4b) and DP1 (Figure 4c).

Changes in telomere biology control gene stability and cell lifespan to inhibit and promote malignant tumors. In the present invention, the present inventors measured telomerase activity in HCCR- 1 or DP1 -transfected RKO cells. As expected, the cancer protein HCCR-1 increased the level of telomerase activity. On the other hand, DP1 transfection decreases telomerase activity in wild-type RKO cells as well as HCCR- 1 -transfected RKO cells (Fig. 4D). These results show that the tumor suppressor candidate DP1 significantly reduces the telomerase activity increased by HCCR-1 in RKO cells.

As can be seen from the above configuration, the results of the present invention indicate that DP1 acts as a tumor suppressor in colon cancer. These results indicate that overexpressed HCCR-1 interacts with DP1 to inhibit the putative tumor suppressor gene DP1, thereby inducing colon cancer tumorigenesis. New tumorigenic pathway and new tumor suppressor DP1 is a target for developing a treatment strategy for colon cancer and may be a useful biomarker for early diagnosis of colon cancer or prognosis of treatment outcome.

1 shows the relationship between HCCR-1 and DP1. (a) is a mapping of the HCCR-1 binding domain of DP1. Six GST fusion constructs, full-DP1p, DP1p1-165, DP1p1-60, DP1p61-185, DP1p61-165, and DP1p121-185 were prepared using DP1 cDNA as template DNA. GST pull-down analysis shows that the four GST-DP1 constructs, pull-DP1, DP1p1-165, DP1p61-185 and DP1p61-165 bind to HCCR-1, but not DP1p1-60 and DP1p121-185. The DP1 binding site of HCCR-1 is depicted as residues 61-120. The hatched box represents the putative transmembrane domain (TMD) and the black box represents the B2-DP1-HVA22 domain of DP1. (b) shows the protein interaction of HCCR-1. HCCR-1 binds to DP1 (top panel). Co-immunoprecipitation was performed from transfected HEK 293 cells producing HCCR-1 and DP1 proteins. Immunoprecipitation was performed with anti-V5 mAb or anti-Myc mAb. Proteins in the pellet were detected with anti-Myc and anti-V5 mAb, respectively. DP1 bound to HCCR-1 (bottom panel). (c) It shows the intracellular location of HCCR-1 in HEK 293 cells. (d) Shows the co-localization of HCCR-1 and DP1 in RKO colorectal cancer cells. It is the intracellular location of HCCR-1 (top panel) and its binding protein, DP1 (bottom panel). The cDNA construct was tagged with HCCR-1 and DP1 as V5 and c-Myc, respectively. VDAC1 (Voltage Dependant Anion Channel 1) was used as a mitochondrial marker.
2 is an expression pattern of HCCR-1 or DP1 in human tissues, cell lines and serum. (a) Comparison of HCCR- 1 mRNA expression in colon cancer cell lines (RKO and HT-29), and fresh primary colon cancer tissues and their corresponding normal tissues. N represents normal colon tissue and C represents primary colon cancer tissue. Human beta actin cDNA was used as a control probe (bottom panel). (b) Immunostaining for HCCR-1 expression in human colon cancer (left panel) and normal colon tissue (right panel), original magnification x 200. (c) Cut-off value determination. ROC curve for 100 people. Although high sensitivity can be obtained by 5.77 μg/ml, the present inventors used 6 μg/ml as a cut-off for practical convenience. (d) DP-1 in colon cancer cell lines, and fresh primary colon cancer tissues and their corresponding normal tissues mRNA expression comparison. (e,f) DP1 expression in several normal human and cancer tissues. Normal 12 lane multiple tissue Northern blot (e) or human cancer cell line multiple Northern blot (f ) was probed with radioisotope labeled DP1 cDNA (top panel) or human beta actin cDNA control probe (bottom panel).
3 is a diagram showing the role of DP1 as a tumor suppressor. (a,b) shows the frequency of LOH around the DP1 gene in primary colorectal cancer. (a) is a representative example of LOH around the DP1 gene. Arrows indicate alleles representing LOH. D5S346 is located within the DP1 gene, and D5S409 and D5S519 are located adjacent to and far from DP1, respectively. (b) is the LOH profile of the colon cancer of 21 investigated. Each box represents an individual colon cancer. Closed box is LOH positive; The open box is LOH negative; N, you are informative; *, indicates microsetlide instability. The LOH frequency was calculated by the following equation. Number of LOH + number of cases/informative cases. (c) It shows the growth inhibitory effect of DP1 in RKO colorectal cancer cells. It is the survival rate of RKO cells after transfection with DP1, pcDNA3.1 vector alone or DP1, respectively. The data are the number of viable cells grown for 10 days in culture and represent the mean±SD of 3 crystals. (d) It is the inhibitory effect of DP1 on transfected colorectal cancer cells. The survival rate of RKO cells after DP1, pcDNA3.1 vector only, DP1, HCCR-1, or co-transfection. (e) Transfection with DP1 cDNA induces apoptosis in colon cancer cells. Electrophoretic analysis of DNAs from wild-type RKO cells, vector only transfected cells, HCCR-1 transfected cells, co-transfected cells or DP1 transfected cells. Cells transfected with the vector or gene were cultured for 3-, 5-, 7- and 9-day. DNAs were analyzed by staining with ethidium bromide on a 2% agarose gel. (f) Analysis of HCCR-1- or DP1-transfected RKO cells using Annexin V-FITC and PI. It is a dot blot of pcDNA3.1, HCCR-1, HCCR-1 and DP1, and DP1-transfected RKO cells. The figure shows surviving (R1; lower left quadrant), annexin V-FITC positive early epitosis (R2; lower right quadrant), and late apoptosis or necrosis (R3; upper right quadrant) cells. (g) It shows the UVC effect on the cell cycle profile phase. Wild type cells, HCCR-1-transfected cells, cotransfected cells or DP1-transfected RKO cells were exposed to UVC of ^{0, 50 or 100 J/m 2, respectively.} After 24 hours of exposure, the treated cells were analyzed according to the method described in the Examples.
4 shows molecular alterations in HCCR- 1 -or DP1 -transfected colorectal cancer cells. (a) p53, p21 ^WAF1 , MDM2 and bax protein expression. Immunoblot analysis of cell lysate from wild type RKO cells, HCCR-1-transfected RKO cells (clone A and B), and DP1-transfected RKO cells (clone A and B) was performed. The same blots were incubated with anti-human mouse beta-actin antibody. (b,c) immunoprecipitation assay. P53 bound to HCCR-1 (b) or DP1 (c), respectively. HEK 293 cells were transfected with pcDNA3.1 encoding p53 protein with pcDNA3.1 encoding HCCR-1-V5-His fusion protein (b) or DP1-Myc-His fusion protein (c), respectively. RKO cells were used as control cells. (d) Determination of telomerase activity. Human telomerase-positive 293 cells, RNase (+RNase) treated 293 cells as a telomerase negative control, wild type RKO cells, cells transfected with HCCR-1 or DP1 were analyzed by telomerase PCR ELISA I did. Assays were performed according to the kit protocol with the same amount of extract as 1 x 10 ^{3 cells.} Telomerase activity in 293 cells used as a positive control was destroyed by pretreatment with RNase. Results are average absorbance values from 4 separate experiments (mean and 95% confidence interval).

Hereinafter, the present invention will be described in more detail through non-limiting examples.

[Example]

Human normal and tumor tissues used in the present invention were obtained in the course of surgery. All patients individually expressed consent to this study. The use of tissue samples has been approved by the hospital's ethics committee. Human Embryonic Kidney (HEK) 293, RKO (ATCC No.; DRL-2577) and HT-29 cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA). Cells were cultured in minimal essential medium Eagle (MEM) with 10% FBS.

Example 1: design of expression vector and DNA Transfection

The expression vector containing the coding region of HCCR-1 or DP1 was designed as follows. First, a Sal I fragment was isolated from pCEV-LAC, a prokaryotic expression vector containing HCCR-1 or DP1 cDNA. Next, pcDNA3.1-V5-His (Invitrogen, CA) or pcDNA3.1-Myc-His was treated with Xho I to prepare a terminal compatible with Sal I. The Sal I fragment containing the coding region of HCCR-1 or DP1 was inserted into Xho I-treated pcDNA3.1. Lipofectamine 2000 (Gibco BRL, Rockville, MD) was used to insert the HCCR- 1 or DP1 expression vector into HEK 293 or RKO cells. Briefly 2 x 10 ⁵ cells were seeded in 60 -mm tissue culture dishes (Costar, Cambridge, MA). After overnight culture in a humidified 5% CO ₂ incubator, cells were treated with 150 ml lipofectamine-DNA complex containing 15 ml lipofectamine and 5 mg of DNA. After 10 hours of incubation, the cells were divided into a ratio of 1:5 and cultured on complete medium containing G418 (Gibco BRL, Gaithersburg, MD). Each cell was selected for resistance to 0.6 mg/ml G418. Selected transfectants were cloned at 0.5 cells per well, maintained in medium containing 0.4 mg/ml G418, and HCCR- 1 or DP1 expression screened by Western blot analysis. Another group of cells were transfected only with the expression vector pcDNA3.1 for use as an experimental control and similarly selected for G418 resistance.

Example 2: Yeast two-hybrid screening and beta-galactosidase analysis

The MATCHMAKER LexA two-hybrid system was used to identify proteins from the human fetal brain MATCHMAKER cDNA library capable of binding to the HCCR-1 fusion protein (Clontech, Palo Alto, CA). All experiments were performed in yeast strain EGY48 transformed with p8op-lacZ (Clontech) expressing lacZ and leu as reporters. The present inventors inserted the HCCR-1 cDNA fragment into a yeast two-hybrid vector (pLexA) (Clontech) containing a LexA DNA-binding domain. Yeast cells expressing LexA-HCCR-1 were transformed with human fetal brain cDNA library (Invitrogen) expressing the B42AD fusion protein. After library transformation, cells were plated in minimal synthetic dropout non-inducing medium (Sigma) selective for both bait (HCCR-1) and AD/library plasmids to increase the chance of AD fusion protein detection. In order to confirm the interaction between HCCR-1 and the binding protein DP1, plasmids expressing HCCR-1 and DP1 were co-transformed into yeast cells. Beta was performed by putting a transformant leaf Rica play-galactosidase during Days filtered lift bunseokreul Trp ^-, Leu ^-, His ^- the PIP expressing HCCR-1 and DP1 on the plate. We used yeast mating assay to eliminate false positive interactions.

Example 3: Simultaneous-transfection and immunoprecipitation

Cells were transfected with pcDNA3.1 (Invitrogen, Carlsbad, CA) encoding the HCCR- 1 -V5-His (Invitrogen) fusion protein and DP1-Myc-His (Invitrogen). After 48 hours, the cells were collected and lysed buffer (20 mM Hepes, pH 7.2, 1% Triton X-100, 1% sodium deoxycholate, 0.2% SDS, 150 mM). NaCl, 1 mM Na ₃ VO ₄ , 1 mM NaF, 10% glycerol, 10 mg/ml leupeptin, 10 mg/ml aprotinin, 1 mM phenymethylsulfonyl fluoride). The lysate was pre-cleaned with preimmune serum (mouse) and protein A-Sepharose at 4° C. for 30 minutes. Protein concentration was determined using a BCA protein assay reagent kit (PIERCE, Rockford, IL) using bovine serum albumin as a standard. Precleaned cell lysate aliquot (1 mg) was added to 1:500 dilution of anti-V5 (Invitrogen) or 1:250 dilution of anti-Myc (Invitrogen) mAb and 1:1 slurry of Protein A- in 40 ml PBS. Sepharose beads were incubated for 16 hours at 4°C. Collect the immune complexes by centrifugation (2,000 g at 4°C for 5 minutes), and buffer (20 mM Tris, pH 7.5, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 2 mM) Na ₃ VO ₄ , 10% glycerol and 1% Nonidet P-40), washed 4 times, SDS-PAGE and anti-Myc of 1:1000 dilution in Tris-buffered saline (TBS) or 1:3000 dilution of anti- Western blot was performed with the V5 antibody.

Example 4: GST fusion protein expression and pull-down experiment

The GST fusion protein was expressed and an extract was prepared as recommended by the manufacturer (Amersham Pharmacia Biotech). Escherichia containing GST, GST-DP1 deletion mutants coli (strain BL21) extract with 30 µl of glutathione-sepharose and 500 µl of Lysis buffer (1 mM phenylmethylsulfonyl fluoride, 5 mM dithiothreitol, 1 µg/ml leupeptin, 1 µg/ml aprotinin, and 1.5 µg/ml pepstatin) 20 mM Tris-HCl [pH 6.8], 50 mM NaCl, 0.1 mM EDTA, 0.1% Triton X-100) was incubated at 4° C. for 12 hours. Fusion proteins bound to Sepharose beads were quantified by Coomassie blue staining on SDS polyacrylamide gel. The fusion protein-bound beads were washed 3 times with 500 μl of TEN buffer (20 mM Tris HCl [pH 7.4], 0.1 mM EDTA, 100 mM NaCl). For the GST pull-down experiment, the fusion protein bound to the beads was incubated at 4° C. for 12 hours with a protein from HEK 293 whole cell extract expressing HCCR-1. The beads were washed with NETN buffer (0.5% Nonidet P-40, 0.1 mM EDTA, 20 mM Tris HCl [pH 7.4], 300 mM NaCl), and 30 μl of SDS sample buffer (75 mM Tris-HCl, pH 6.8, 0.5% glycerol, 1% SDS, 4% mercaptoethanol, 0.01% bromophenol blue) and heated for 3 minutes before separation on a 10% SDS-polyacrylamide gel. The separated protein was subjected to Western analysis.

Example 5: Intracellular location

Intracellular fractionation of cells was performed according to mitochondrial extraction (Pierce, Rockford, IL). Briefly, cells were collected by centrifugation at 850 g for 2 minutes, and the pellet was suspended with 800 µl of reagent A, and then incubated on ice for exactly 2 minutes. 10 μl of reagent B was added to the suspended solution and incubated with vortexing for 5 minutes each minute at maximum speed. 800 [mu]l of reagent C was added to the solution and inverted several times for mixing. The solution was centrifuged at 700 xg at 4°C for 10 minutes, and the pellet was used for crude nuclear fractionation. The supernatant was centrifuged at 12,000 xg at 4°C for 15 minutes, and transferred to a new tube for protoplasmic fractionation. The pellet was washed with 500 μl of reagent C and used for the isolated mitochondrial fraction. Each fraction was quantified with the BCA Protein Analysis Reagent Kit (Pierce). The heated extract in the sample buffer was subjected to electrophoresis on SDS-PAGE and transferred to nitrocellulose by standard methods. Block the membrane with 5% skim milk in Tris buffer saline (TBS; pH 7.4) with 0.05% Tween 20 and anti-V5 (Invitrogen, Carlsbad, CA), anti-c-Myc ( Santa Cruz Biotechnology, Santa Cruz, CA) or anti-VDAC1 (Santa Cruz Biotechnology). After washing with 0.05% Tween 20 in TBS, the immunoblot was incubated with horseradish-peroxidase-conjugated goat-anti-mouse-IgG, or goat-anti-rabbit-IgG, and proteins were incubated according to the manufacturer's instructions (Pierce). Saw using increased chemilaminesense.

Example 6: Northern blot analysis

Northern blot analysis was performed to investigate HCCR-1 or DP1 expression in several human tissues using either the HCCR-1 cDNA or the 534-bp partial DP1 cDNA. The mRNA expression level was quantified by comparison with the expression level of beta-actin.

Example 7: Antibody production and purification

167 ^th to 360 ^th The C-terminal portion of human HCCR-1 cDNA encoding a polypeptide, which is amino acid residues, was cloned into a pMAL ^TM -p2X (New England Biolabs, MA) vector, and it was expressed in E. coli strain BL21 DE3. Recombinant HCCR-1 protein was purified from bacteria using amylose resin. The fusion protein was digested with factor Xa proteinase, and the resulting HCCR-1 protein was used to prepare mAbs. For mAb production, 2 month-old Balb/c mice were immunized with HCCR-1 antigen prepared as an emulsion with Freund's adjuvant. Immune responses were monitored by Western blot using ELISA and antisera obtained from animals at different times of immunization. Mice exhibiting high immunoreactivity to the antigen were selected for the production of hybridoma cell lines. Three days after the pre-fusion boost with antigen, the mice were sacrificed, and lymphocytes from spleen were fused with SP 2/0 Maeroma cells using polyethylene glycol. In order to select the surviving fusion clones producing antibodies, the fused cells were grown for 10 days in Dulbecco's minimum essential medium containing hypoxanthine-minopterin-hymidine. The medium containing the surviving fusion clones was tested for ELISA and Western blot to confirm specific immunoreactivity to the antigen. Those fusion clones selected from primary screening were more single-cell subclones using the same approach and final hybrid cell lines were selected from single cell subclones. To generate ascites, HCCR-1-specific mAb-producing hemibridoma cells were injected ^{5×10 6} cells per mouse into the peritoneal cavity of prestatin-treated male balb/c mice. After 10 days, ascites were extracted from mice and centrifuged to remove cells and blood debris. Then, the ascites were incubated with sodium dextran sulfate and CaCl _{2 to precipitate lipoproteins.} Certain IgG was precipitated with 50% ammonium sulfate in the ascites.

Example 8: Detection of HCCR-1 in serum by sandwich ELISA

HCCR-1 levels were measured using a sandwich ELISA. _Passage -diluted purified HCCR-1 167-360 was coated on a 96-well maxisorp microtiter plate in 100 μl of 10 mM phosphate buffer and cultured. A standard curve was made from the passage 20 to the purified HCCR-1 _{167- 360} dilution of the 320 ng / ml range. Calibration curves were prepared using 20, 40, 80, 160, and 320 ng/ml HCCR-1 _{167-360 protein.} The microtiter plate was then saturated with PBS containing 2 mg/ml BSA. Selected pairs of antibodies (JE33 and I87 mAbs) that recognize a different epitope for each HCCR-1 molecule were used to formulate a sandwich ELISA. Purified primary mAb JE33 (100 ng/ml per well) was prepared in PBS containing 0.1% BSA and 0.05% Tween 20. Serum samples diluted 1:100 were analyzed in duplicate. 100 µl of the sample was plated on the wells and washed with PBS containing 0.05% Tween 20. After washing, a peroxidase-attached detector mAb (I87) prepared at 2 μg/ml was reacted at 37° C. for 1 hour. The absorbance at 492 nm was measured with a spectrometer. A standard curve was created and reference samples were included in each test run. The HCCR-1 concentration in each serum sample was calculated by reference to a standard curve and expressed as HCCR-1 nanograms per ml of serum. The reference range was obtained by receiver operating characteristic (ROC) curve analysis: a value of 6 μg/ml was used as the cut-off for colorectal cancer. CA19-9 was measured by a two-step sandwich immunoradiametric analysis using a commercially available kit (CA19-9 IRMA kit, Fujirebio Diagnostics, Inc., Malvern, PA).

Example 9: Subjects for ELISA

During the study period from December 2003 to December 2004, a total of 100 people enrolled in the Catholic University of Korea in Gangnam St. Mary's Hospital. 50 (average age = 55 years old, ranging from 25 to 78 years old) were histologically proven or clinically diagnosed with colorectal cancer, and 50 were normal volunteers (average age = 46 years old, 18 years old) -82 years old). None of those patients had other tumors or active lung disease. Clinical pathologic factors were extracted from initial surgical pathology data. Stage was determined according to the Tumor-Nodule-Metastases (TNM) Stage System of Colorectal Cancer according to the American Joint Community for Cancer. Serum was obtained prior to treatment from 50 patients newly diagnosed with colorectal cancer. Analysis was performed for all patients and controls with the individual consent of the study. The use of blood samples was approved by the Foundation's Ethics Committee.

Example 10: Epitope mapping by analysis of time-dependent trypsin-treated section patterns

MBP-HCCR-1 fusion protein (10 μM) Mic Each mAb (5 μM) was mixed in 3 μl of PBS, and only MBP-HCCR-1 of the same concentration was also prepared in PBS. Digestion reaction was initiated by adding 8 μM trypsin to the pre-culture sample for 30 minutes. At a specific time point, 0.5 μM of each solution was extracted to complete the cleavage reaction with a matrix solution [matrix, alpha-cyano-4-hydroxycinnamic acid (CHCA) 0.5% TFA in 70% acetonitrile] containing [0104]. The finished samples were either loaded directly into the MALDI plate or kept at -20°C until further analysis. Amersham Ettan pro MALDI-TOF (Amersham Bioscience, Piscataway, NJ) was used to collect spectra of the treated sections. All spectra collected were obtained in the cationic reflector mode. 500, typically with 400-500 power per spectrum Sat was added. For spectral analysis, theoretical trypsin fragments of MBP-HCCR-1 were calculated by ProteinProspector (www.prospector.ucsf.edu).

Example 11: Sample for LOH

Frozen tissues were randomly collected from 21 patients with colon cancer who underwent surgery at Dankook University Hospital in Cheonan, Korea. The entire process of tissue collection and genetic analysis was performed under the approval of the evaluation committee of the Catholic University of Korea, Gangnam St. Mary's Hospital. For each case, the tumor cell-rich area (more than 60% tumor cells) was microdissected and the excised cells (50-80 cells/μL) were 50mM Tris-HCl (pH 8.5), 0.5% tween 20, and 0.2 mg/ml. Maintained under digestion buffer containing proteinase K. The microdissection buffer was incubated at 50° C. for 18-24 hours and purified using a DNA purification kit (Solgent, Korea).

Example 12: Analysis of LOH around DP1 with microsatellite marker

The surrounding three microsatellite locations were investigated for LOH using the following markers on chromosome 5, D5S519, D5S409, and D5S346. D5S346 (112.2 Mb) is located in the DP1 gene, D5S409 (102.7 Mb) is located above 9.5 Mb of DP1, and D5S519 (149.9 Mb) is located below 37.7 Mb of DP1. Microdissected genetic DNA was amplified with 1 μCi 32p-labeled dCTP in a 10 μl PCR mixture. 32 rounds of amplification reactions were carried out under the following conditions: 94°C for 30 seconds, 55°C for 1 minute and 72°C for 1 minute. The amplified product was diluted with a gel-loading buffer containing 95% formaldehyde, heated for 5 minutes, and completely denatured, followed by ice cooling. Samples were electrophoresed on a 6% modified acrylamide gel and visualized by autorography.

Example 13: Analysis of LOH

The signal intensity of each allele band was measured using a densitometer. LOH was measured only in the Informative (heterologous) locus. Non-informational locus means that both alleles of the locus are homologous and cannot be used to interpret LOH. When the band intensity ratio between the two alleles of tumor cells differed by more than 20% than that of normal cells, it was determined that this tumor had LOH in the microsatellite locus investigated. The non-informational locus after the LOH-positive locus was determined as LOH positive, and the non-informational locus after the LOH-negative locus was interpreted as LOH-negative.

Example 14: DNA modification by sodium bisulfate

90 [mu]l of the tissue lysate was denatured with 10 [mu]l of 3 M NaOH at 37[deg.] C. for 15 minutes. The mixture was modified with 1040 μl of 2.3 M sodium bisulfate and 60 μl of 10 mM hydroquinone for 12 hours at 50° C. and purified using Wizard DNA purification resin (Promega, Madison, WI) according to the manufacturer's instructions. The modified DNA was finally precipitated by adding 2.5 volume of 100% ethanol and 1 µl of salmon testis DNA (5 mg/ml, Sigma-Aldrich, St. Louis, MO). The pellet was washed with 70% ethanol, dried and dissolved in 35 μl of 5 mM Tris buffer (pH 8.0). To avoid repeated freezing and thawing, 1 µl aliquot of the modified DNA solution was placed in a PCR tube and stored at -20°C.

Example 15: Methylation and demethylation-specific PCR and bisulfate sequencing

The MSP primer set was designed using MethPrimer software (http://www.urogene.org/methprimer/) and sequence redundancy was checked by BLAST search. Primers are as follows: DP1MF: 5'-AGTTTTTTTCGTGTAGGAATCGGTC-3' (SEQ ID NO: 4), DP1MR: 5'-CAATCGCCGCTCCAATC-TATCCG-3' (SEQ ID NO: 5), DP1UF: 5'-AGTTTTTTTGTGTAGGAATTGGTT-3' (sequence Number:6), DP1UR: 5'-CAATCACCACTCCAATCTATCCA-3' (SEQ ID NO:7). Genetic DNA (CpGenome Universal Methylated DNA, Chemicon, Temecula, CA, USA) universally methylated by DNA methylase was used as the methylation control DNA. PCR DNA amplified by the universal primer (5'-CCGACTCGAGNN NNNNATGTGG-3') (SEQ ID NO: 8) was used as the demethylation control DNA. Universal PCR was performed as follows: 1 cycle of denaturation at 94°C for 3 minutes, 10 cycles of 94°C for 1.5 minutes, 30°C for 2.5 minutes, 72°C for 3 minutes and 30 cycles of 94°C for 1 minute, It is an extension of 1.5 minutes at 62°C, 2 minutes at 72°C and 8 minutes at 72°C. After confirming the methylation-specific and demethylation-specific amplification, 21 pairs of normal and colon cancer were analyzed under these conditions.

Example 16: DNA fragmentation analysis

HCCR- 1 -or DP1 -transfected colorectal cancer cells, cells transfected with pcDNA3.1 alone, and wild-type cells, respectively, were collected after culturing for 3, 5, 7 and 9 days. The cells were disrupted and overnight at 48° C. in a disruption buffer containing 100 μg/ml of Proteinase K [10 mM Tris-HCl (pH 7.4), 10 mM EDTA, 10 mM NaCl, and 0.5% SDS] . 1/5 volume of 5 M NaCl and an equal volume of isopropyl alcohol were added to precipitate the DNA. The DNA pellet was redissolved in TE buffer [10 mM Tris-HCl (pH 7.8) and 1 mM EDTA] and treated with 0.1 mg/ml of RNase A at 37° C. for 1 hour. For each sample, 10 μg DNA was fractionated on a 2% agarose gel, stained with ethidium bromide and visualized with UV light.

Example 17: Detection of apoptosis by Annexin V/PI staining

Cells were washed with cold PBS and stained with Annexin V/PI according to Aubry, JP et al. Cytometry 37, 197-204 (1999) and analyzed on a BRYTE HS flow cytometer (Bio-rad, Herts, England). . Briefly, after washing with PBS, 1 × 10 ⁷ cells were diluted with 10 μl of 10 × binding buffer [100 mM HEPES (pH 7.4), 1.5 M NaCl, 50 mM KCl, 10 mM MgCl ₂ and 18 mM CaCl _2] Afterwards, 1 μl Annexin V-conjugate and 10 μl PI were added according to the manufacturer's instructions for an epitosis kit (TACSTM Annexin V-FITC, Trevigen, Inc., Gaithersburg, MD). The cell suspension was incubated for 15 minutes in a dark room at room temperature, and 400 μl of 1 × binding buffer was added before flow cytometric analysis.

Example 18: Telomerase activity assay

Telomerase activity was determined using a telomerase PCR-ELISA kit (Boehringer Mannheim, Germany). A negative control was provided for human 293 cells pretreated with RNase.

Example 19: Statistical Analysis

Serum levels (log values, μg/ml) of HCCR-1 in both groups and at each stage of the pathologic factor were expressed as mean±S.D and compared using Wilcoxon rank sum test and Kruskal-Wallis test. To determine the positive response, we used a value of 37 U/ml as the cut-off for CA19-9 and the cut-off value for HCCR-1 was determined by ROC curve analysis. Positive reaction rates for CA19-9 and HCCR-1 were expressed as marginal rates and compared by McNemar test. The overall accuracy and sensitivity of HCCR-1 were measured and their 95% confidence interval was indicated. CA19-9 and HCCR-1 positive rates in each step were compared by McNemar test. Two-tailed, 0.05 level of significance, and SAS release 8.1 were used for all statistical analyses.

<110> KIM, HYUN KEE <120> COMPOSITION FOR DIAGNOSING AND/OR TREATING CANCER AND THE KIT <160> 8 <170> KopatentIn 1.71 <210> 1 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> 169-187 amino acid residue of HCCR-1 <400> 1 His Phe Trp Thr Pro Lys Gln Gln Thr Asp Phe Leu Asp Ile Tyr His 1 5 10 15 Ala Phe Arg <210> 2 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> 291-310 amino acid residue of HCCR-1 <400> 2 Ala Lys Leu Gly Ile Gly Gln Leu Thr Ala Gln Glu Val Lys Ser Ala 1 5 10 15 Cys Tyr Leu Arg 20 <210> 3 <211> 185 <212> PRT <213> Artificial Sequence <220> <223> DP1 <400> 3 Met Arg Glu Arg Phe Asp Arg Phe Leu His Glu Lys Asn Cys Met Thr 1 5 10 15 Asp Leu Leu Ala Lys Leu Glu Ala Lys Thr Gly Ala Asn Arg Ser Phe 20 25 30 Ile Ala Leu Gly Val Ile Gly Leu Val Ala Leu Tyr Leu Val Phe Gly 35 40 45 Tyr Gly Ala Ser Leu Leu Cys Asn Leu Ile Gly Phe Gly Tyr Pro Ala 50 55 60 Tyr Ile Ser Ile Lys Ala Ile Glu Ser Pro Asn Lys Glu Asp Asp Thr 65 70 75 80 Gln Trp Leu Thr Tyr Trp Val Val Tyr Gly Val Phe Ser Ile Ala Glu 85 90 95 Phe Phe Ser Asp Ile Phe Leu Ser Trp Phe Pro Phe Tyr Tyr Met Leu 100 105 110 Lys Cys Gly Phe Leu Leu Trp Cys Met Ala Pro Ser Pro Ser Asn Gly 115 120 125 Ala Glu Leu Leu Tyr Lys Arg Ile Ile Arg Pro Phe Phe Leu Lys His 130 135 140 Glu Ser Gln Met Asp Ser Val Val Lys Asp Leu Lys Asp Lys Ala Lys 145 150 155 160 Glu Thr Ala Asp Ala Ile Thr Lys Glu Ala Lys Lys Ala Thr Val Asn 165 170 175 Leu Leu Gly Glu Glu Lys Lys Ser Thr 180 185 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Primer DP1MF <400> 4 agtttttttc gtgtaggaat cggtc 25 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer DP1MR <400> 5 caatcgccgc tccaatctat ccg 23 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer DP1UF <400> 6 agtttttttg tgtaggaatt ggtt 24 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Primer DP1UR <400> 7 caatcaccac tccaatctat cca 23 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Universal primer <400> 8 ccgactcgag nnnnnnatgt gg 22

Claims (1)

A composition for treating colorectal cancer comprising the amino acid of SEQ ID NO: 3 as an active ingredient.

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