Abstract:
The present invention provides a new additional identification of a gene related to cancer expression and a diagnostic kit using the same.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application claims priority under 35 U.S.C. 119 based upon Japanese Patent Application Serial No. 2005-001033, filed on Jan. 5, 2005. The entire disclosure of the aforesaid application is incorporated herein by reference. 
     FIELD OF THE INVENTION 
     The present invention relates to a cancer-specific gene and a diagnostic kit using the same, and further relates to a method for using the diagnostic kit. 
     BACKGROUND OF THE INVENTION 
     The most crucial challenge in measures against cancer is the early detection of cancer. Particularly, early detection is important for cancers originating from the upper part of the large intestine since they cause only limited subjective symptoms and the medical condition may likely to be in its advanced stage by the time of discovery. 
     Traditional measures against large intestine cancer include screening by the fecal occult blood test, diagnosis by serum markers such as CEA or CA19-9, and diagnosis during a course of treatment. However, the positive rates of these methods are high only for advanced cancers and extremely low for early cancers, making accurate diagnosis difficult in their early stages. 
     Meanwhile, a biological diagnostic method using a cancer tissue-specific protein marker is suggested as a method allowing simple and reliable early diagnosis of malignancy. This method can be performed on a broad range of asymptomatic subjects since it does not require a large-scale facility and causes small burdens for the subject. For instance, Japanese Patent Application Publication No. H07-51065 discloses a usage of glycoprotein 39 as a tumor marker. 
     In addition, International Patent Application Publication No. WO/2004/018679 describes a technology regarding a cancer diagnostic kit using CENP-A. Sugata, N., et al., “Human CENP-H multimers colocalize with CENP-A and CENP-C at active centromere-kinetochore complexes,”  Hum. Mol. Genet ., vol. 9, no. 19, 2000, pp. 2919-2926 discloses the sequences of human CENP-H protein and its corresponding encoding nucleotide which correspond to SEQ ID No: 1 and SEQ ID No: 2 of the present application. Sugata et al. also discloses the biochemical characterization and the localization of CENP-H protein suggesting its role in cell cycle progression. 
     WO 03/104426 discloses CENP-E with additional background information relating to CENP-A, B, C, and D. Also, WO 03/104426 discloses a method to detect the abnormal amount of CENP-E protein in biopsied tissue for diagnosis of predisposition or actual clinical symptoms of cancer and the kits for detecting the presence of aberrant CENP-E protein expression. 
     However, cancer expression cannot be thoroughly verified by the technology described in the above JP-A-H7-51065 alone and a plurality of means must be used to ensure positive identifications. 
     Considering the above situation, the purpose of the present invention is to provide a further identification of a gene related to cancer expression and a diagnostic kit using the same. 
     SUMMARY OF THE INVENTION 
     Considering the above situation, the present invention employs specific means described below. 
     A first means is a polynucleotide as in one of the following (a)-(c): 
     (a) a polynucleotide consisting of a base sequence as in SEQ ID NO: 1 or a complementary base sequence thereof; (b) a polynucleotide consisting of a base sequence having at least 70% homology with the base sequence as in SEQ ID NO: 1 or the complementary base sequence thereof; and (c) a polynucleotide coding a protein consisting of an amino-acid sequence as in SEQ ID NO: 2, or another amino-acid sequence defined by the amino-acid sequence having one or several amino acid deletions, substitutions or additions, wherein the polynucleotide is a marker for detecting cancer. 
     The polynucleotide in this means may greatly contribute to a cancer diagnosis when used as a marker since the polynucleotide has been discovered to exhibit a high expression in cancer tissues. The homology with the base sequence as in SEQ ID NO: 1 is preferably equal to or greater than 70%, more preferably equal to or greater than 80%, and even more preferably equal to or greater than 90%. 
     A second means is a cancer diagnostic kit comprising a primer consisting of a base sequence as in SEQ ID NO: 3. 
     A third means is a cancer diagnostic kit comprising a primer consisting of a base sequence as in SEQ ID NO: 4. 
     A fourth means is a cancer diagnostic kit comprising a primer set consisting of the primers as in SEQ ID NOS: 3 and 4. Each of these cancer diagnostic kits may be used to diagnose rectal cancer or colon cancer. 
     A fifth means is a method comprising the steps of: measuring an expression level of a protein consisting of an amino-acid sequence as in SEQ ID NO: 2 for each of two collected cells; and determining whether or not the ratio between the measured expression levels is equal to or great than 1.7. In this case, one of the two samples is collected from a non-cancer tissue; the other sample is collected from, for example, a tissue suspected to be a cancer tissue; and if the expression level ratios of the two samples are different, the subject from whom the samples were collected may be determined to be at a high risk of cancer. 
     Preferably for this means, the step of measuring the expression level of the protein is performed with the western blot, wherein one of the two collected cells is a cell in a non-cancer tissue, wherein one of the two collected cells is a cell in a cancer tissue. 
     As described above, a gene related to cancer expression may be newly identified, and a diagnostic kit using the same may be provided. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a diagram showing western blot results for CENP-H; 
         FIG. 2  is a diagram showing western blot results for hMis12; 
         FIG. 3  is a diagram showing the results of staining cross-sectional areas of rectal cancer tissues and their respective nearby non-rectal cancer tissues with anti-human CENP-H polyclonal antibody; 
         FIG. 4  is a diagram showing analysis results on amounts of CENP-H mRNA in each of the surfaces of rectal cancer tissues and normal tissues using RT-PCR and real-time quantitative PCR; and 
         FIG. 5  is a diagram showing analysis results on amounts of CENP-H mRNA in each of the surfaces of rectal cancer tissues and normal tissues using RT-PCR and real-time quantitative PCR. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     One embodiment of the present invention will be described below. 
     Tissue Collection 
     Body tissues were collected with a surgical method from 15 patients with early-stage colorectal cancer. The tissues were taken from cancer tissue (hereinafter referred to as “cancer tissue”) and tissue at a part 5-10 cm away from the cancer tissue (hereinafter referred to as “non-cancer tissue”), respectively. The collected tissues were immersed in liquid nitrogen and stored at −80° C. 
     Protein Extraction 
     The cryonically-preserved tissues were then placed into lysis buffer (7 M urea, 2 M thiourea, 2% 3-[(3-Cholamidopropyl)Dimethylammonio]-1-Propanesulfonate, 0.1 M DTT, 2% IPG buffer (made by Amersham Pharmacia Biotech), 40 mM Tris), lysed using polytron homogenizer (made by Kinematica), and centrifuged at 10,000 g and 4° C. for 1 hour to collect supernatant and extract proteins. 
     Immunoblot 
     The proteins were blotted to polyvinylidene fluoride membranes (made by Millipore) in a tank transfer device (made by Bio-Rad) and the membranes were then blocked with phosphate buffered saline (PBS) containing 5% skim milk. Next, 1:5000 diluted rabbit anti-CENP-H antibody, 1:100 diluted rabbit anti-hMis12 antibody and 1:500 diluted goat anti-β-actin antibody, each placed in the blocking buffer, were used as a primary antibody; and 1:3000 diluted goat anti-rabbit IgG HRP and 1:500 diluted rabbit anti-goat IgG HRP, each placed in the blocking buffer, were used as a secondary antibody. 
     Note that antibodies on the antigen membrane were detected with enhanced chemiluminescence detection reagent (made by Amersham Pharmacia Biotech). Also the intensity of each band was measured with an NIH image. 
     PCR and Real-Time Quantitative PCR 
     TotalRNA was extracted from the cancer tissue and the non-cancer tissue, respectively, using RNeasy Mini Kit (made by Qiagen). Also cDNA was synthesized from each extracted totalRNA, respectively, using a 1st Strand cDNA Synthesis Kit for RT-PCR (made by Roche). 
     Then each cDNA obtained by this synthesis was used as a template to amplify the cDNA of CEMP-H with PCR. In the PCR, a primer comprising a base sequence as in SEQ ID NO: 3 or 4 were used as the forward and reverse primer, respectively, and cDNA of GAPDH or β-actin were amplified as the controls. 
     Subsequently cDNA real-time quantitative PCR for CENP-H was performed in a LIGHTCYCLER®capillary. For the PCR reaction mixture, 3.0 mM of MgCl 2 , 0.5 μM of the primer as in SEQ ID NO: 3 and 0.5 μM of the primer as in SEQ ID NO: 4 were added to LIGHTCYCLER® DNA Master SYBR Green I (FastStart Taq DNA polymerase, dNTP, buffer, SYBR Green I), and the procedure was conducted within a total of 2.0 μl. 
     LIGHTCYCLER® software version 3.3 (made by Roche) was then used for analysis. 
     Immunohistochemical Staining Method 
     The frozen tissue sections were dried on a glass slide and fixed in 4° C. acetone. The tissues were then washed with PBS 3 times and blocked with the blocking buffer (10% fetal bovine serum/PBS) for 1 hour. 
     The sample was incubated for 1 hour in 3% bovine serum albumin/PBS using one or both of 1:2000 diluted rabbit anti-CENP-H antibody and 1:1000 diluted anti-human CENP-A monoclonal antibody. After washing with PBS, the sample was incubated for 1 hour with 1:1000 diluted ALEXA FLUOR® 488- or 594-bound goat anti-rabbit anti-mouse IgG secondary antibody (made by Molecular Probes) and/or ALEXA FLUOR® 594-bound goat anti-mouse IgG secondary antibody. 
     DNAs were counterstained using DAPI III Counterstain (made by Vysis). The sample was observed with a fluorescence microscope (made by Leica QFISH). The tissue sections were stained with hematoxylin for 30 minutes for HE staining, dried over 100% ethanol and xylene and encapsulated with Permount. 
     Results 
       FIG. 1  shows results of the western blot. As shown in  FIG. 1 , CENP-H was highly expressed in the cancer tissue in any of the 15 cases. Particularly, a ratio between the non-cancer tissue and the cancer tissue CENP-H expressions was 1.7-9.6, indicating a large difference between these two kinds of tissues. For another centromere protein hMis12, on the other hand, no notable difference was discovered between the cancer tissue and the non-cancer tissue. (See  FIG. 2 , in which tissues with the same case number as in  FIG. 1  are identical with those in  FIG. 1 ). 
     Next, in order to verify that CENP-H is expressed in the cancer cell, but not in stromal cells, cross-sectional areas of colorectal cancer tissues and nearby non-cancer tissues were stained with an anti-human CENP-H polyclonal antibody. The results are shown in  FIG. 3 . Note that  FIG. 3(   a ) shows an HE-stained image of the cancer tissue;  FIGS. 3(   b ), ( c ) and ( d ) show a CENP-H antibody immunostained image of the cancer tissue;  FIG. 3(   e ) shows an HE-stained image of the non-cancer tissue; and  FIG. 3(   f ) shows a CENP-H-stained image of the non-cancer tissue. 
     As a result, it was confirmed that CENP-H existed as small patchy points in cell nuclei at positions coinciding with the centromeres in a similar manner to that of other centromere proteins such as CENP-A and CENP-C. It was also confirmed that the CENP-H had been increased both in number and size in the cancer tissues ( FIGS. 3(   c ) and ( d )) compared to the non-cancer tissue ( FIG. 3(   f )). It should be noted that the stained CENP-H was verified not in the stromal cells, but in the cancer tissue epithelia. Also the present experiment was conducted on. various tissue sections and all the results were similar to each other. 
     Accordingly, it was confirmed that CENP-H was expressed in cancer cells. 
     Subsequently, in order to verify that the CENP-H overexpression was a result of its increase due to transcription, amounts of mRNA of CENP-H in the colorectal cancer tissues and the non-cancer tissues were analyzed, respectively, using RT-PCR and real-time quantitative PCR. The results are shown in  FIGS. 4 and 5 . 
     As shown in  FIG. 4 , an expression level of the CENP-H mRNA in the cancer tissues is far more increased than in the non-cancer tissues. Furthermore, it was discovered that the expression level of the CENP-H mRNA indicated a strong correlation with the CENP-H expression ratio between the non-cancer tissues and the cancer tissues illustrated in  FIG. 1 . As a control, when confirmation was done pertaining to GAPDH, there was no significant difference in the expression level of the CENP-H mRNA between the cancer tissues and the non-cancer tissues. 
       FIG. 5  illustrates a comparison of the mRNA expression levels shown in  FIG. 4  between the non-cancer tissues and the cancer tissues using StatView statistical analysis software. According to  FIG. 5 , the CENP-H mRNA expression level in the cancer tissues (Cancer) was 5 times higher than that of the non-cancer tissues (Normal). In this way, the presence of cancer can be also verified by examining the CENP-H expression level. 
     INDUSTRIAL AVAILABILITY 
     Thus according to the present invention, a gene related to cancer expression may be newly identified, and a diagnostic kit using the same may be also provided.