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From the Department of Forensic Medicine,* Division of Human Genetics, and the Departments of Pathology and Surgery,¶ Kurume University School of Medicine, Kurume; the Department of Pathology, St. Mary’s Hospital, Kurume; and the Department of Physiology, University of Occupational and Environmental Health, Kitakyushu, Japan
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Abstract |
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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.7-10 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,11,12 most of which harbor mutations in the tumor suppressor adenomatous polyposis coli (APC) gene.13-15 He and colleagues16 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.17 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.18 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,18 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.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Recombinant human Mina53 was expressed in Escherichia coli and isolated as described previously.18 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.
Other Antibodies
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-ß-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.
Plasmids
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)18 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)19 to produce pCAGGS/hmina53(465). pEGFP/hmina53(465) expressing green fluorescent protein (GFP)-Mina53 fusion protein was described previously.18
Cell Culture and Transfections
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.18 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.18 SW620 cells were transfected with siRNA basically as described previously.18 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.
Western Blotting and Indirect Immunofluorescence Staining
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-ß-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-ß-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.
Tissues for Immunostaining
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.20
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
.
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Deparaffinized sections of 10% formalin-fixed, paraffin-embedded colon tumor tissues were immunostained by the streptavidin-biotin complex immunoperoxidase method.21 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.
Evaluation of Immunohistochemical Staining
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.
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Results |
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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 ß-actin expression did not differ between lanes (Figure 1A
, lanes 1 to 3, bottom), which confirmed that similar amounts of total protein were electrophoresed.
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, lane 1). HL60 cells are terminally differentiated by treatment with TPA in which the c-myc expression level is reduced.22,23
We showed previously that the expression of mina53 is reduced after treatment of HL60 cells with TPA using anti-Mina53 polyclonal antibody.18
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 ß-actin expression (Figure 1A
, lanes 1 and 2). These results indicate that the monoclonal antibody M532 recognizes specifically Mina53 protein. Expression of Mina53 in Colon Tumor Cell Lines
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.18
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.18
Mina53 Is Involved in Proliferation of Colon Tumor Cells
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.18
These results suggest that Mina53 is involved in proliferation of colon tumor cells.
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We previously demonstrated that mina53 is a direct Myc target gene.18
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.
Elevated Expression of Mina53 in Human Colon Tumor Tissues Detected by Western Blotting Analysis
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 ß-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.
Elevated Expression of Mina53 in Colon Tumor Tissues Detected by Immunohistochemical Analysis
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 staining. Staining is found mainly in nuclei (Figure 3, E and F)
with dotted staining in nucleoli, a pattern similar to those obtained from the colon tumor cell line (Figure 1C)
. There was a lack of specific nuclear staining when the first antibody was omitted (Figure 3D)
or when the section was incubated with the primary antibody in the presence of an excess amount of recombinant Mina53 protein (not shown).
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. In addition, tumor cells that had penetrated into lymphatic vessels in deeper layers of the colon also showed strong immunoreactivity to Mina53 antibody, whereas surrounding nonneoplastic cells were not stained (Figure 4B)
. M532 antibody also stained deeply invading tumors (Figure 4C)
and isolated poorly differentiated adenocarcinoma cells in fibrous stroma (Figure 4D)
, whereas nonneoplastic cells surrounding these tumor cells were not stained. These results suggest that Mina53 is highly expressed in tumors with invasive and metastatic potential.
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. Figure 4
, F and H, shows expression of Mina53 and c-Myc, respectively, in the tumor, but not in nonneoplastic cells surrounding these tumor cells. The germinal center showed little staining by both anti-Mina53 M532 and anti c-Myc antibodies, although the germinal center is known to contain many well-proliferating cells. Ki-67, a biomarker for cell proliferation, was highly expressed in the germinal center (Figure 4G)
, confirming that the germinal center contains many proliferating cells. These results showed that Mina53 staining patterns correlate well with those of c-Myc in colon tumor tissues. Mina53 Expression by Pathological Grade of Colon Cancer
Mina53 was detected in specimens from all 23 patients with colon adenocarcinoma and 1 patient with adenoma. A summary of the immunohistochemical staining is presented in Table 1
. Mina53 was expressed in all pathological grades of colon cancer. Well (Figure 5A)
, moderately (Figure 3B)
, and poorly (Figure 3F)
differentiated adenocarcinomas were all markedly stained for Mina53 with average staining indexes of 2.28, 1.78, and 1.85, respectively (Table 1)
. These results suggest that there was no obvious correlation between the pathological grade of cancer and staining intensity, although there was a slight tendency for well-differentiated adenocarcinomas to show higher staining intensity than poorly and moderately differentiated ones (Table 1)
. In addition, Mina53 was highly expressed in the adenoma (Figure 5C)
, suggesting that the elevated expression of Mina53 is a general and relatively early event in colon carcinogenesis. As described above, staining was confined to the nucleus in all cases (Figure 3, E and F)
.
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Serial sections were also stained with anti-Ki-67 monoclonal antibody (MIB 1) to compare the staining pattern with that of Mina53. As reported before,24-26
anti-Ki-67 monoclonal antibody stained colon tumor tissues intensely (Figures 3C, 4G, and 5B)
. In most cases both antibodies against Ki-67 and Mina53 stained tumor cells similarly, as shown in Figure 3
(B and C), Figure 4
(F and G), Figure 5
(A and B), and Table 1
.
When the percentages of tumor cells in a microscopic field stained for both Ki-67 and Mina53 were compared, differences were found between the two antibodies. In the majority of the cases, the percentage of Mina53-expressing cells was higher than that of Ki-67 (Figure 5, A and B)
. In a few cases, the percentage of Mina53-expressing cells was similar to that of Ki-67 (Table 1)
.
In one case (patient 3), the staining pattern was rather complex. Sections contained three different types of cancer. Two areas with poorly differentiated adenocarcinoma cells and mucinous adenocarcinoma cells showed similarly high percentages of positive cells for both Mina53 and Ki-67 (Table 1)
. In the third area, however, the tumor had a morphology similar to acinar cell carcinoma of the pancreas and was very weakly stained by Mina53 antibody but was strongly stained by Ki-67 antibody, although the percentage of positive cells was low as compared to the former two areas (Table 1)
. In the case of the adenoma (patient 24), almost all cells in the neoplastic areas were intensely stained by M532 antibody, whereas fewer cells were stained by anti-Ki-67 (Figure 5, C and D)
. Although there was one exception (patient 3), the results described above suggest that the percentage of Mina53-expressing cells is characteristically higher than that of Ki-67-expressing cells.
Comparison between Staining Patterns of Mina53 and Ki-67 in Nonneoplastic Colon Tissues
In some areas of nonneoplastic colon tissues, antibody against Ki-67 stained cells intensely, whereas M532 antibody stained those cells only weakly. Ki-67 staining was observed in nuclei of cells in the lower third of the crypts, the zone of active cell proliferation (Figure 5F)
. The staining intensity in nonneoplastic crypts did not differ significantly from cells found in the tumor areas (Figure 5, B and F)
. On the other hand, M532 antibody only faintly stained nuclei of these cells, and the intensity was far weaker than cells found in the tumor areas (Figure 5, A and E)
.
A lymphoid germinal center, which contains cells with high mitotic activity, was intensely stained by Ki-67 antibody (Figure 4G)
, but was rarely stained by M532 antibody (Figure 4F)
as described before. These results suggest that Ki-67 is generally expressed in proliferating cells, whereas Mina53 is not always expressed in all proliferating cells.
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Discussion |
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We used antibody M532 to study the expression of Mina53 in colon tumor cell lines and colon tumor tissues. The two colon tumor cell lines showed prominent expression of Mina53. Our results showed that almost all colon tumor tissues examined exhibited elevated expression of Mina53. Mina53 was expressed in colon cancer of all pathological grades, well, moderately, and poorly differentiated, while most nonneoplastic cells showed little or no staining for Mina53. The elevated expression of Mina53 in tumor cells was observed irrespective of the location of the cells; that is, tumor cells in primary neoplasia, invading tumors, and tumors in lymphatic vessels as well as isolated tumor cells were prominently stained for Mina53 protein. We also observed that a higher percentage of tumor cells expressed Mina53 than Ki-67, a frequently used cell proliferation marker. Antibody to Ki-67 strongly stained tumor cells as well as some well-proliferating normal cells, including cells in the lower crypts and lymphoid germinal centers. On the other hand, antibody to Mina53 rarely stained cells in nonneoplastic areas. These results indicate that high expression of Mina53 can be regarded as a characteristic feature of colon cancer. Thus, the expression of Mina53 can be used as a marker for colon cancer and may be helpful in finding tumor cells that have invaded normal tissues. In addition, Mina53 staining may also serve as an additional prognostic marker. For example, detection of Mina53 staining in cells of lymph vessels may allow the selection of patients for systemic therapies.
In addition to the elevated expression of Mina53 in all pathological grades of colon cancer, the elevated expression of Mina53 was also observed in the adenoma. These results suggest that the elevated expression of Mina53 is a general event that occurs early in colon carcinogenesis and continues during the tumor progression. Thus, the high expression of Mina53 in colon cancer may be because of molecular impairment that occurs as an early event during carcinogenesis. With this assumption, impairment of the Wnt/APC/ß-catenin pathway appears to be a good candidate. Somatic mutations in the APC gene were found in more than 60% of colon cancers and were suggested to be early events in colon carcinogenesis.14,15 The main function of APC protein is thought to be the regulation of free ß-catenin in concert with glycogen synthase kinase-3ß (GSK-3ß) and axin proteins.27 Mutations of ß-catenin were also found in some colon tumors lacking APC mutations.28,29 Loss of APC function, inactivation of axin,30 or activating ß-catenin mutations31 results in cellular accumulation of ß-catenin, which, when translocated to the nucleus, serves as an activator of T-cell factor (Tcf)-dependent transcription leading to increased expression of several specific target genes.32 He and colleagues16 identified c-myc as a target of the APC pathway and demonstrated that c-myc is induced by loss of function of the APC gene or by overexpression of ß-catenin. Therefore, inactivating mutations of APC or axin or activating mutations of ß-catenin would result in overexpression of c-myc. Mina53 staining patterns correlate well with those of c-Myc in colon tumor tissues. Suppression of c-myc expression by the specific siRNA in colon tumor cells reduced expression of mina53. These results suggest that c-Myc induced expression of Mina53 in colon tumor cells. Thus, overexpression of c-myc would subsequently result in the elevated expression of Mina53. It seems therefore that mina53 is a downstream, indirect target of the APC/ß-catenin pathway, which has been implicated in colon carcinogenesis.
The expression pattern of Mina53 was compared with that of Ki-67, a widely used biomarker of cell proliferation. The percentage of Mina53-expressing cells was characteristically higher than that of Ki-67-expressing cells in most tumor tissues (Table 1)
. This may be because of the fact that mina53 is a Myc target gene. C-myc is expressed continuously in all phases of the cell cycle in proliferating cells,33,34
whereas Ki-67 is preferentially expressed in proliferating cells in late G1, S, M, and G2 phases.35,36
Because Mina53 is induced by c-Myc, it is reasonable that Mina53 is more widely expressed than Ki-67 in tumor tissues.
Cells in the crypts, which have been shown to grow well, were intensely stained by anti-Ki-67 antibody, but only weakly by anti-Mina53 antibody. Lymphoid germinal centers that contain nonneoplastic but proliferating cells were weakly stained by Mina53 antibody but strongly stained by Ki-67 antibody. Our previous18 and present studies show that Mina53 is an important factor for cell proliferation in cultured cell lines, human cervical carcinoma HeLa cells, a rat fibroblast cell, and colon cancer cells, all of which highly express c-myc. Thus, it is possible that Mina53 may play a role in cell proliferation only in some restricted types of cells, for example in cells with high expression of c-Myc. Alternatively, normal cells in the crypts and lymphoid germinal centers may require smaller amounts of Mina53 for cell proliferation or these cells may express a protein with a similar function as Mina53 protein. Either way, because tumor cells were intensely stained by anti-Mina53 antibody as compared to nonneoplastic cells in vivo, Mina53 may have some functions in carcinogenesis.
We also observed a lack of expression of c-Myc in lymphoid germinal centers, the staining pattern being similar to that observed for Mina53, which is consistent with our conclusion that mina53 is a Myc target gene. These observations, although surprising because c-Myc is known to be involved in cell proliferation, were similar to the results obtained recently by Klein and colleagues37 who demonstrated a lack of c-Myc expression in tonsillar germinal centers. They reported that expression of the other Myc family members, such as N-Myc or L-Myc, did not appear to compensate for the absence of c-Myc in germinal center B cells, because these cells also lacked expression of mRNA for these genes. Our observation that Myc target gene mina53 was poorly expressed in lymphoid germinal centers suggests that there is little Myc activity there, and is consistent with the observations of Klein and colleagues37 that N-Myc and L-Myc in addition to c-Myc were not expressed in cells in lymphoid germinal centers.
Another important issue that arises from this study is a treatment strategy for patients with colon cancers. Pharmacological strategies that target inactivation of oncogenes for the treatment of cancer are in development because activation of oncogenes has been shown to be associated with carcinogenesis. However, long-term use of medicines that are designed to inactivate oncogenes would be expected to produce serious toxicities because they also disrupt critical signal pathways in normal cells.38 The myc family oncogenes are thought to be central regulators of cell growth, and studies have shown that they are critical for normal biological processes, including organ development and regeneration.7-9,39,40 C-myc controls the expression of various genes, and specific Myc target genes are thought to be the actual players in each Myc function. This therefore calls for a thorough study of novel Myc target genes. Knowledge of the functions of these genes may help to develop medicines that are designed to inactivate specifically the functions involved in carcinogenesis with little effect on normal physiological processes. Although the specific role of Mina53 protein is not yet fully characterized, a specific siRNA duplex for human mina53 severely suppressed proliferation of colon cancer cells, suggesting that mina53 is a candidate target gene for colon cancer therapy.
In conclusion, we generated a specific anti-human Mina53 monoclonal antibody that will aid further studies on this novel Myc target gene. The antibody demonstrated increased expression of Mina53 in colon tumors as compared to nonneoplastic colon tissues. Our results suggest that the expression of Mina53 is an early event and a characteristic feature in colon cancer and that Mina53 may therefore play some role in colon carcinogenesis. We suggest that Mina53 can be used as a marker for colon cancer and that Mina53 may be exploited as a target for treatment of colon cancer.
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Acknowledgements |
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Footnotes |
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Supported by grants-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan; and from the Novartis Foundation (Japan) for the Promotion of Science.
Accepted for publication September 25, 2003.
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