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Increased Expression of a Myc Target Gene Mina53 in Human Colon Cancer

2004; Elsevier BV; Volume: 164; Issue: 1 Linguagem: Inglês

10.1016/s0002-9440(10)63111-2

1525-2191

Kwesi Teye, Makoto Tsuneoka, Nobuyuki Arima, Yoshiro Koda, Yasuhiro Nakamura, Yoichi Ueta, Kazuo Shirouzu, Hiroshi Kimurâ,

Mechanisms of cancer metastasis

Mina53 is a novel Myc target gene that we previously demonstrated to be involved in cell proliferation. We studied, here, the expression of Mina53 in colon cancer to examine its possible role in carcinogenesis. We generated a specific monoclonal anti-human Mina53 antibody and found that colon tumor cell lines expressed Mina53 highly. We also found that expression of Mina53 was elevated in colon tumor tissues by immunoblotting analysis. Tissue sections of 23 surgical cases of adenocarcinoma and 1 case of adenoma were stained immunohistochemically, and the expression of Mina53 was found to be elevated in all of the adenocarcinomas compared to adjacent nonneoplastic tissues, which showed little staining. Deeply invading tumors as well as tumors that have invaded lymphatic vessels showed strong immunoreactivity against anti-Mina53 antibody. Mina53 was expressed in all pathological grades of cancer as well as in the adenoma. Staining patterns of Ki-67, a biomarker for cell proliferation, were similar to those of Mina53 in most cases, but the percentage of tumor cells stained by anti-Mina53 was higher. Although anti-Ki-67 antibody strongly stained some well-proliferating nonneoplastic cells including cells in the deeper part of the crypts and in lymphoid germinal centers, antibody to Mina53 rarely stained those cells. Suppression of mina53 expression severely suppressed proliferation of colon tumor cells in vitro. Together, our results indicate that the elevated expression of Mina53 is a characteristic feature in colon cancer, one that may have therapeutic applications. Mina53 is a novel Myc target gene that we previously demonstrated to be involved in cell proliferation. We studied, here, the expression of Mina53 in colon cancer to examine its possible role in carcinogenesis. We generated a specific monoclonal anti-human Mina53 antibody and found that colon tumor cell lines expressed Mina53 highly. We also found that expression of Mina53 was elevated in colon tumor tissues by immunoblotting analysis. Tissue sections of 23 surgical cases of adenocarcinoma and 1 case of adenoma were stained immunohistochemically, and the expression of Mina53 was found to be elevated in all of the adenocarcinomas compared to adjacent nonneoplastic tissues, which showed little staining. Deeply invading tumors as well as tumors that have invaded lymphatic vessels showed strong immunoreactivity against anti-Mina53 antibody. Mina53 was expressed in all pathological grades of cancer as well as in the adenoma. Staining patterns of Ki-67, a biomarker for cell proliferation, were similar to those of Mina53 in most cases, but the percentage of tumor cells stained by anti-Mina53 was higher. Although anti-Ki-67 antibody strongly stained some well-proliferating nonneoplastic cells including cells in the deeper part of the crypts and in lymphoid germinal centers, antibody to Mina53 rarely stained those cells. Suppression of mina53 expression severely suppressed proliferation of colon tumor cells in vitro. Together, our results indicate that the elevated expression of Mina53 is a characteristic feature in colon cancer, one that may have therapeutic applications. Colon cancer is among the most frequent neoplasms in the world. Epithelial crypts of the colon are presumed to be the sites from which most neoplastic cells arise. It is currently thought that most colon cancers develop from pre-existing adenomas, although some may emerge de novo.1Fearon ER Vogelstein B A genetic model for colon tumorigenesis.Cell. 1990; 61: 759-767Abstract Full Text PDF PubMed Scopus (9839) Google Scholar, 2Kuramoto S Oohara T Flat early cancers of the large intestine.Cancer. 1989; 64: 950-955Crossref PubMed Scopus (177) Google Scholar, 3Bedenne L Faivre J Boutron MC Piard F Cauvin JM Hillon P Adenoma-carcinoma sequence or "de novo" carcinogenesis? A study of adenomatous remnants in a population-based series of large bowel cancers.Cancer. 1992; 69: 883-888Crossref PubMed Scopus (197) Google Scholar, 4Wada R Matsukuma S Abe H Kuwabara N Suda K Arakawa A Kitamura S Histopathological studies of superficial-type early colon carcinoma.Cancer. 1996; 77: 44-50Crossref PubMed Scopus (81) Google Scholar Most colon cancers are known to progress through a series of gradual histological changes from premalignant and malignant stages to the metastatic state.1Fearon ER Vogelstein B A genetic model for colon tumorigenesis.Cell. 1990; 61: 759-767Abstract Full Text PDF PubMed Scopus (9839) Google Scholar, 5Vogelstein B Kinzler KW The multistep nature of cancer.Trends Genet. 1993; 9: 138-141Abstract Full Text PDF PubMed Scopus (1501) Google Scholar, 6Kinzler KW Vogelstein B Lessons from hereditary colon cancer.Cell. 1996; 87: 159-170Abstract Full Text Full Text PDF PubMed Scopus (4230) Google Scholar Many investigations have been directed toward uncovering changes in the gene structure, gene expression, or the activity of gene products associated with colon cancer. Most studies have shown that loss of function of tumor suppressor genes as well as activation and abnormal expression of oncogenes are responsible for carcinogenesis. The members of the myc proto-oncogene family, c-, L-, and N-myc, are thought to be central regulators of cell growth, and deregulated expression of myc is associated with many cancers.7Henriksson M Lüscher B Proteins of the Myc network: essential regulators of cell growth and differentiation.Adv Cancer Res. 1996; : 109-182Crossref PubMed Google Scholar, 8Grandori C Cowley SM James LP Eisenman RN The Myc/Max/Mad network and the transcriptional control of cell behavior.Annu Rev Cell Dev Biol. 2000; 16: 653-699Crossref PubMed Scopus (1012) Google Scholar, 9Lüscher B Function and regulation of the transcription factors of the Myc/Max/Mad network.Gene. 2001; 277: 1-14Crossref PubMed Scopus (202) Google Scholar, 10Nesbit C Tersakk J Prochownik E Myc oncogenes and human neoplastic disease.Oncogene. 1999; 18: 3004-3016Crossref PubMed Scopus (953) Google Scholar C-myc is one of the well-studied oncogenes and its expression is associated with cell proliferation and is down-regulated in quiescent and differentiated cells. Studies have shown that c-myc is overexpressed in most human colon cancers,11Stewart J Evan G Watson J Sikora K Detection of the c-myc oncogene product in colonic polyps and carcinomas.Br J Cancer. 1986; 53: 1-6Crossref PubMed Scopus (129) Google Scholar, 12Sikora K Chan S Evan G Gabra H Markham N Stewart J Watson J C-myc oncogene expression in colon cancer.Cancer. 1987; 59: 1289-1295Crossref PubMed Scopus (160) Google Scholar most of which harbor mutations in the tumor suppressor adenomatous polyposis coli (APC) gene.13Cottrell S Bicknell D Kaklamanis L Bodmer WF Molecular analysis of APC mutations in familial adenomatous polyposis and sporadic colon carcinomas.Lancet. 1992; 340: 626-630Abstract PubMed Scopus (211) Google Scholar, 14Miyoshi Y Nagase H Ando H Horii A Ichii S Nakatsuru S Aoki T Miki Y Mori T Nakamura Y Somatic mutations of the APC gene in colon tumors: mutation cluster region in the APC gene.Hum Mol Genet. 1992; 1: 229-233Crossref PubMed Scopus (860) Google Scholar, 15Powell SM Zilz N Beazer-Barclay Y Bryan TM Hamilton SR Thibodeau SN Vogelstein B Kinzler KW APC mutations occur early during colon tumorigenesis.Nature. 1992; 359: 235-237Crossref PubMed Scopus (1641) Google Scholar He and colleagues16He TC Sparks AB Rago C Hermeking H Zawel L da Costa LT Morin PJ Vogelstein B Kinzler KW Identification of c-MYC as a target of the APC pathway.Science. 1998; 281: 1509-1512Crossref PubMed Scopus (4026) Google Scholar provided a molecular framework for understanding the previously enigmatic overexpression of c-myc in colon cancers by identifying c-myc as a target of the APC pathway. They demonstrated that c-myc is either induced by loss of function of the APC gene or suppressed by the functional APC gene product. Despite intensive efforts to investigate the role(s) of c-myc in carcinogenesis, the mechanisms by which deregulation of c-myc gene expression contributes to carcinogenesis are still not fully resolved, and many aspects are still enigmatic.17Lutz W Leon J Eilers M Contributions of Myc to tumorigenesis.Biochim Biophys Acta. 2002; 1602: 61-71PubMed Google Scholar C-myc is a multifunctional gene, and its functions include cell division, cell growth, and apoptosis. C-myc appears to control the expression of several genes that mediate each of the above functions, some of which may contribute to carcinogenesis. Functional information about expression patterns of novel genes controlled by c-myc may therefore contribute to a better understanding of carcinogenesis induced by c-myc. Recently we identified a novel gene, mina53, whose expression was demonstrated to be directly induced by c-myc.18Tsuneoka M Koda Y Soejima M Teye K Kimura H A novel Myc target gene, mina53, that is involved in cell proliferation.J Biol Chem. 2002; 277: 35450-35459Crossref PubMed Scopus (105) Google Scholar The mina53 gene encodes a 53-kd protein that is localized in the nucleus with part of the protein concentrated in the nucleolus. Specific inhibition of mina53 expression by the RNA interference (RNAi) method severely suppressed cell proliferation,18Tsuneoka M Koda Y Soejima M Teye K Kimura H A novel Myc target gene, mina53, that is involved in cell proliferation.J Biol Chem. 2002; 277: 35450-35459Crossref PubMed Scopus (105) Google Scholar suggesting that Mina53 may be implicated in carcinogenesis. To address the question of whether Mina53 is expressed in human cancer and to evaluate its possible role in carcinogenesis, we generated a specific monoclonal antibody against human Mina53 protein and examined the expression of Mina53 in colon tumor cell lines and in surgically resected colon tumor tissues. Here we show that Mina53 is highly expressed in colon cancer and that Mina53 is involved in proliferation of colon tumor cells in vitro. Our results suggest that Mina53 may have a functional role in colon carcinogenesis and may be of use as a marker for colon cancer. Recombinant human Mina53 was expressed in Escherichia coli and isolated as described previously.18Tsuneoka M Koda Y Soejima M Teye K Kimura H A novel Myc target gene, mina53, that is involved in cell proliferation.J Biol Chem. 2002; 277: 35450-35459Crossref PubMed Scopus (105) Google Scholar BALB/c mice were immunized via the sole with purified Mina53 emulsified in monophosphoryl-Lipid A + Trehalose dicorynomycolate adjuvant (Sigma-Aldrich Fine Chemicals, St. Louis, MO) and boosted after 2 weeks. Lymphocytes were isolated from lymph nodes of the hind limb and fused with mouse F01 myeloma cells using polyethylene glycol following the standard method. Cells were cultured along with cells from the thymus in 96-well plates in RPM1 1640 medium (Sigma) supplemented with 20% fetal calf serum, hypoxanthine/aminopterin/thymidine (ICN Biochemicals, Aurora, OH), and 5% Briclone hybridoma cloning medium (Archport, Dublin, Ireland). Approximately 2 weeks after fusion, the hybridoma culture media were screened for anti-Mina53 antibody activity using microtiter plates coated with recombinant Mina53. Cells in positive wells were cloned and culture media consistently positive after recloning were tested for specificity of the antibodies by Western blotting using HL60 and HeLa cell extracts. Hybridomas secreting specific antibodies were isotyped using a Zymed mouse MonoAB ID/SP kit (Zymed Laboratories, South San Francisco, CA) following the manufacturer's instructions. One hybridoma secreting an IgG2a antibody, clone M532, was selected for this study. The antibody was produced as ascites fluid in prestane-preinjected mice and purified using a DE52 anion exchange resin (Whatman International, Kent, UK) after 50% ammonium sulfate fractionation. The IgG purity was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Coomassie brilliant blue staining. Rabbit polyclonal anti-nucleolin (sc-13057) and anti-c-Myc (N-262) antibodies and goat anti-rabbit IgG-horseradish peroxidase (HRP) (Santa Cruz Biotechnology, Santa Cruz, CA), Alexa 488-conjugated anti-mouse IgG (Molecular Probes, Eugene, OR), goat anti-mouse IgG-HRP and Cy3-conjugated anti-rabbit IgG (Zymed), anti-β-actin monoclonal antibody (AC-15) (Sigma), mouse monoclonal anti-Ki-67 (MIB-1) (DAKO, Glostrup, Denmark), and biotinylated rabbit anti-mouse IgG and biotinylated goat anti-rabbit IgG (Nichirei, Tokyo, Japan) were purchased. cDNA for human mina53 was amplified by polymerase chain reaction with 5′-GCCCAAGCTTACCATGCCAAAGAAAGCAAAGCCTAC-3′, adding a HindIII site before the initiation methionine, and 5′-GCCCAAGCTTCTAGACTACTTGAATTAAACATTC-3′, adding a HindIII site after the stop codon as primers from pT/hmina53(465)18Tsuneoka M Koda Y Soejima M Teye K Kimura H A novel Myc target gene, mina53, that is involved in cell proliferation.J Biol Chem. 2002; 277: 35450-35459Crossref PubMed Scopus (105) Google Scholar The amplified 1.4-kb fragment was cloned into a pCAGGS mammalian expression vector (containing a chimeric promoter consisting of chicken actin and a CMV promoter)19Sunaga S Maki K Komagata Y Ikuta K Miyazaki JI Efficient removal of lox-P flanked DNA sequences in a gene-targeted locus by transient expression of Cre recombinase in fertilized eggs.Mol Reprod Dev. 1997; 46: 109-113Crossref PubMed Scopus (30) Google Scholar to produce pCAGGS/hmina53(465). pEGFP/hmina53(465) expressing green fluorescent protein (GFP)-Mina53 fusion protein was described previously.18Tsuneoka M Koda Y Soejima M Teye K Kimura H A novel Myc target gene, mina53, that is involved in cell proliferation.J Biol Chem. 2002; 277: 35450-35459Crossref PubMed Scopus (105) Google Scholar Two human colon tumor cell lines, SW620 and HT-29, and the human cervical carcinoma cell line HeLa were maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. The human promyelocytic leukemia cell line HL60 was maintained in RPMI 1640 medium supplemented with 20% fetal calf serum. HL60 cells were cultured in the presence or absence of 10 nmol/L of phorbol 2-myristate 13-acetate (TPA) for 24 hours. HeLa cells were transfected with plasmids pCAGGS/hmina53(465) or pEGFP/hmina53(465) using FuGENE 6 transfection reagent (Roche Diagnostics, Indianapolis, IN) to express Mina53 or GFP-Mina53 fusion protein, respectively. A small interference RNA molecule (siRNA) targeting human c-myc and a control siRNA (catalog no. 4604; Ambion Austin, TX) were purchased. The siRNA sequences targeting human mina53 and rat mina53 were described previously.18Tsuneoka M Koda Y Soejima M Teye K Kimura H A novel Myc target gene, mina53, that is involved in cell proliferation.J Biol Chem. 2002; 277: 35450-35459Crossref PubMed Scopus (105) Google Scholar The siRNA duplex specific for rat mina53 differed by five nucleotides of a 19-nucleotide sequence targeting human mina53 and has been shown not to affect mina53 expression in human HeLa cells.18Tsuneoka M Koda Y Soejima M Teye K Kimura H A novel Myc target gene, mina53, that is involved in cell proliferation.J Biol Chem. 2002; 277: 35450-35459Crossref PubMed Scopus (105) Google Scholar SW620 cells were transfected with siRNA basically as described previously.18Tsuneoka M Koda Y Soejima M Teye K Kimura H A novel Myc target gene, mina53, that is involved in cell proliferation.J Biol Chem. 2002; 277: 35450-35459Crossref PubMed Scopus (105) Google Scholar Briefly, 24 hours before transfection, cells were transferred to a 12-well plate coated with collagen type I (Asahi Technoglass, Chiba, Japan). Transfection was performed with 100 pmol of siRNA per well using OligofectAMINE (Invitrogen, Tokyo, Japan), according to their instruction, except that SW620 cells were cultured for 36 hours for transfection without serum. The number of cells was counted at specific time intervals. For Western blotting, cells were collected by treatment with trypsin and ethylenediaminetetraacetic acid in phosphate-buffered saline (PBS) and washed with PBS. Cells were then suspended in 0.125 of mol/L Tris-HCl buffer, pH 6.8, containing 3% sodium dodecyl sulfate, 50 mmol/L dithiothreitol, and 20% glycerol and boiled for 10 minutes before separation on a gradient sodium dodecyl sulfate-polyacrylamide gel (4 to 20%). Proteins were transferred to a polyvinylidene difluoride microporous membrane (Millipore, Bedford, MA), and nonspecific binding sites were blocked with 1% skim milk in PBS. After treatment with mouse monoclonal anti-Mina53 and HRP-conjugated goat anti-mouse IgG, signals were detected using an enhanced chemiluminescence Western blotting detection reagent system (Amersham Biosciences, Buckinghamshire, UK). The membrane was reprobed with a monoclonal anti-β-actin antibody, as described above, after treatment with a stripping buffer (Pierce, Rockford, IL). Some membranes were also treated with rabbit polyclonal anti-c-Myc and HRP-conjugated goat anti-rabbit IgG antibodies. Human colon tumor specimens were surgically resected from four patients. Tissues were sliced into small pieces in PBS (10 μl per mg wet tissue). Solubilization buffer (0.125 mmol/L Tris-HCl, pH 6.8, 3% sodium dodecyl sulfate, 100 mmol/L dithiothreitol) was added (10 μl per mg wet tissue), boiled for 10 minutes, and centrifuged at 14,000 rpm for 10 minutes. The total protein concentration of the supernatant was determined using Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA). Five μg of total protein was electrophoresed and subjected to Western blotting with anti-Mina53, anti-c-Myc, or anti-β-actin antibodies as described above. For indirect immunofluorescence staining, SW620 and HT-29 cells grown on glass coverslips were fixed in methanol for 10 minutes at −20°C. Mouse anti-Mina53 monoclonal and rabbit anti-nucleolin polyclonal antibodies were added and incubated for 120 minutes at 37°C. After three washes with 0.1% skim milk in PBS, Alexa 488-conjugated anti-mouse IgG and Cy3-conjugated anti-rabbit IgG were added, incubated for 120 minutes at 37°C, and washed three times with 0.1% skim milk in PBS. Finally, cells were embedded in Immunon (Thermo Shandon, Pittsburgh, PA) and observed with a fluorescence microscope. Routinely processed formalin-fixed and paraffin-embedded specimens from 24 patients with primary colon neoplasia resected from 1988 to 1996 at the Department of Surgery, Kurume University Hospital, were used.20Fujitani N Liu Y Toda S Shirouzu K Okamura T Kimura H Expression of H type 1 antigen of ABO histo-blood group in normal colon and aberrant expressions of H type 2 and H type 3/4 antigens in colon cancer.Glycoconjugate J. 2000; 17: 331-338Crossref PubMed Scopus (27) Google Scholar The specimens included 23 cases of adenocarcinoma of the colon, in some of which tumor cells had invaded deeply into nonneoplastic tissues or into lymphatic vessels. There was also one case of adenoma. Tissue sections were classified after hematoxylin and eosin (H&E) staining according to the pathological grading system as well, moderately, and poorly differentiated. The characteristics of the tissues are outlined in Table 1.Table 1Pathological Grade of Colon Tumors and Summary of Immunohistochemistry% Stained cancer cellsStaining intensityStaining indexPatientGradeMina53Ki-67Mina53Ki-67Mina53Ki-671Poor84.546.2110.850.462Poor86.363.2332.591.903Poor79.688.9231.592.67*The area contained mucinous adenocarcinoma cells.100.096.3222.001.93†The cancer had a morphology similar to acinar cell carcinoma of the pancreas.0.031.9020.000.644Poor94.252.4231.881.575Poor100.0100232.003.006Moderate94.381.6221.891.637Moderate100.076.2333.002.298Moderate97.587.4231.952.629Moderate100.083.6131.002.5110Moderate92.475.0221.851.5011Moderate79.262.1221.581.2412Moderate89.054.2120.891.0813Moderate100.060.1131.001.8014Moderate95.584.8332.872.5415Well80.172.9221.601.4616Well100.063.5222.001.2717Well93.850.0322.811.0018Well100.0100.0333.003.0019Well92.784.6231.852.5420Well78.774.0231.572.2221Well98.957.0231.981.7122Well92.743.1332.781.2923Well98.380.0332.952.4024Adenoma90.042.6322.700.85* The area contained mucinous adenocarcinoma cells.† The cancer had a morphology similar to acinar cell carcinoma of the pancreas. Open table in a new tab Deparaffinized sections of 10% formalin-fixed, paraffin-embedded colon tumor tissues were immunostained by the streptavidin-biotin complex immunoperoxidase method.21Fujitani N Liu Y Okamura T Kimura H Distribution of H type 1–4 chains of the ABO(H) system in different cell types of human respiratory epithelium.J Histochem Cytochem. 2000; 48: 1649-1656Crossref PubMed Scopus (14) Google Scholar Sections mounted on slides were autoclaved for 20 minutes in 10 mmol/L of sodium citrate buffer, pH 6.0, for antigen retrieval. After pretreatment with 3% H2O2 in PBS and then with 1% skim milk and 5% rabbit serum in PBS, the primary antibody against Mina53 at a final concentration of 3.5 μg/ml in 1% skim milk or anti-Ki-67 antibody (used as recommended by the manufacturer) was reacted with tissues overnight at 4°C in a moist chamber. After three washes with 0.05% Tween 20 in PBS, sections were incubated sequentially with biotinylated rabbit anti-mouse IgG and then with HRP-streptavidin conjugate (Nichirei). Color was developed with 3,3-diaminobenzidine and H2O2 for 4 (Mina53) or 2 (Ki-67) minutes and then a water rinse was used to stop the reaction. After light counterstaining with hematoxylin, the slides were dehydrated, coverslipped, and observed with an Olympus AX80 microscope (Olympus Optical, Tokyo, Japan). A few sections were also stained with rabbit polyclonal anti-c-Myc antibody as described above. Each section was scored on a scale from 0 to 3 by visual observation. The highest staining intensity was scored as 3, the lowest as 1, and no staining at all as 0. For estimation of the percentage of stained cells, images were captured with an Olympus digital camera (Olympus), processed with Photoshop, and printed out. The number of positive cells within representative fields was counted and expressed as the percentage of cells stained. The staining index was calculated as staining intensity multiplied by the average percentage of cells stained. We generated a monoclonal antibody against recombinant human Mina53 protein. The initial screening assays by enzyme-linked immunosorbent assay resulted in a panel of monoclonal antibodies. One of the monoclonal antibodies, M532, recognized a single band with a molecular mass of 53 kd by Western blotting in the human cervical carcinoma cell line HeLa (Figure 1A, lane 1). M532 also recognized increased expression of Mina53 in HeLa cells transfected with a mammalian expression vector harboring mina53 cDNA (Figure 1A, lane 2). When Mina53 fused to green fluorescent protein (GFP) was expressed in HeLa cells, Western blotting analysis using M532 monoclonal antibody yielded a band with an expected molecular mass of 80 kd in addition to the endogenous Mina53 (Figure 1A, lane 3). In this experiment β-actin expression did not differ between lanes (Figure 1A, lanes 1 to 3, bottom), which confirmed that similar amounts of total protein were electrophoresed. M532 also recognized a single band with a molecular mass of 53 kd by Western blotting in human promyelocytic leukemia cell line HL60 (Figure 1B, lane 1). HL60 cells are terminally differentiated by treatment with TPA in which the c-myc expression level is reduced.22Hozumi M Fundamentals of chemotherapy of myeloid leukemia by induction of leukemia cell differentiation.Adv Cancer Res. 1983; 38: 121-169Crossref PubMed Scopus (168) Google Scholar, 23Hickstein DD Back AL Collins SJ Regulation of expression of the CD11b and CD18 subunits of the neutrophil adherence receptor during human myeloid differentiation.J Biol Chem. 1989; 264: 21812-21817Abstract Full Text PDF PubMed Google Scholar We showed previously that the expression of mina53 is reduced after treatment of HL60 cells with TPA using anti-Mina53 polyclonal antibody.18Tsuneoka M Koda Y Soejima M Teye K Kimura H A novel Myc target gene, mina53, that is involved in cell proliferation.J Biol Chem. 2002; 277: 35450-35459Crossref PubMed Scopus (105) Google Scholar Immunoblotting analysis using the M532 monoclonal antibody showed that treatment of cells with TPA reduced the signal of the 53-kd band (Figure 1B, lane 2). The specificity of the reduction was confirmed because TPA treatment did not significantly reduce β-actin expression (Figure 1A, lanes 1 and 2). These results indicate that the monoclonal antibody M532 recognizes specifically Mina53 protein. The expression of Mina53 was examined in two colon tumor cell lines, SW620 and HT-29. Cell lysates were prepared from the cells in the proliferating phase and analyzed by immunoblotting using M532 antibody. The antibody recognized a single band of 53 kd in the two cell lines (Figure 1B, lanes 3 and 4). The results indicate that these cell lines express Mina53 and that antibody M532 specifically recognizes Mina53 protein in colon tumor cell lines with no cross-reactivity with other proteins. The expression level of Mina53 in these cell lines is much higher than that of Mina53 in HL60 cells experimentally reduced by TPA (Figure 1B, lanes 2 to 4). The levels of actin in the two colon tumor cell lines were not higher than that of HL60 cells treated with TPA (Figure 1B, lanes 2 to 4). These results suggest that the two colon tumor cell lines contain a higher level of Mina53 protein than terminally differentiated HL60 cells. We previously demonstrated that Mina53 is localized in the nucleus and also concentrated in the nucleolus in HeLa cells.18Tsuneoka M Koda Y Soejima M Teye K Kimura H A novel Myc target gene, mina53, that is involved in cell proliferation.J Biol Chem. 2002; 277: 35450-35459Crossref PubMed Scopus (105) Google Scholar To investigate the localization of Mina53 in colon tumor cells, we performed double-immunofluorescence staining of cells using monoclonal antibody M532 and anti-nucleolin rabbit antibody. As shown in Figure 1C, M532 antibody stained specifically nuclei in SW620 cells with strong dotted staining in nucleoli that overlapped with the signals for nucleolin. The other cell line HT-29 showed a similar pattern of immunofluorescence staining (not shown). These results indicate that Mina53 locates in the nucleus with concentrated amounts in the nucleolus in the colon tumor cell lines, as we previously demonstrated in HeLa cells.18Tsuneoka M Koda Y Soejima M Teye K Kimura H A novel Myc target gene, mina53, that is involved in cell proliferation.J Biol Chem. 2002; 277: 35450-35459Crossref PubMed Scopus (105) Google Scholar To gain insight into the role of Mina53 in colon tumor cells, we specifically suppressed the expression of Mina53 in a colon tumor cell line SW620 by a specific siRNA for human mina53 (Figure 2A, lane 3). As shown in Figure 2B, reduction of Mina53 expression severely suppressed proliferation of SW620 cells. Treatment of cells with a nonspecific siRNA duplex and a specific siRNA for rat mina53 neither reduced expression of Mina53 nor suppressed proliferation of SW620 cells [Figure 2, A (lanes 1 and 4) and B]. The siRNA duplex specific for rat mina53 differed by five nucleotides of a 19-nucleotide sequence targeting human mina53 and had been shown to affect mina53 expression in rat cells but not in human HeLa cells.18Tsuneoka M Koda Y Soejima M Teye K Kimura H A novel Myc target gene, mina53, that is involved in cell proliferation.J Biol Chem. 2002; 277: 35450-35459Crossref PubMed Scopus (105) Google Scholar These results suggest that Mina53 is involved in proliferation of colon tumor cells. We previously demonstrated that mina53 is a direct Myc target gene.18Tsuneoka M Koda Y Soejima M Teye K Kimura H A novel Myc target gene, mina53, that is involved in cell proliferation.J Biol Chem. 2002; 277: 35450-35459Crossref PubMed Scopus (105) Google Scholar To examine the regulation of mina53 expression by c-Myc in colon tumor cells, the colon tumor cell line SW620 was treated with a specific siRNA for c-myc to suppress the expression of c-Myc. Specific suppression of c-Myc expression resulted in reduction of Mina53 expression (Figure 2A, lane 2). Treatment of cells with nonspecific control siRNAs reduced neither c-Myc nor Mina53 expression (Figure 2A, lanes 1 and 4). These results indicate that the expression of mina53 is regulated by c-Myc in colon tumor cells. Tumor and adjacent nonneoplastic tissues derived from surgical specimens from four patients were analyzed by Western blotting for Mina53 and c-Myc proteins. In three cases, expression of Mina53 was clearly elevated in tumor tissues as compared to their nonneoplastic counterparts (Figure 2C, cases 1, 3, and 4). In these cases, expression of c-Myc was also clearly elevated in the tumor tissues as compared to their nonneoplastic counterparts. In one case in which c-Myc expression was hardly detected in either tumor or nonneoplastic tissues, the expression of Mina53 was only slightly elevated in the tumor tissue as compared to the nonneoplastic tissue (Figure 2C, case 2). The results showed a positive correlation between Mina53 and c-Myc levels in colon cancer. In all cases, the levels of β-actin were not significantly different. These results indicate that Mina53 expression is elevated in colon cancer and closely related to c-Myc expression, which is consistent with our conclusion that Mina53 is a Myc target gene. Monoclonal antibody M532 was used to detect Mina53 protein immunohistochemically in colon tumor tissues. H&E staining was used to demarcate the tumor areas. The section shown in Figure 3A contained moderately differentiated adenocarcinoma. Figure 3B shows marked staining for Mina53 in tumor areas, whereas most nonneoplastic epithelial cells around the tumors showed little s