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Clinical Cancer Research 13, 858, February 1, 2007. doi: 10.1158/1078-0432.CCR-06-1461
© 2007 American Association for Cancer Research
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Human Cancer Biology

NF-Y–Dependent Cyclin B2 Expression in Colorectal Adenocarcinoma

Seon-Hwa Park1, Gyung-Ran Yu1, Woo-Ho Kim4, Woo-Sung Moon2, Jong-Hun Kim3 and Dae-Ghon Kim1

Authors' Affiliations: 1 Division of Gastroenterology and Hepatology, Departments of Internal Medicine, 2 Pathology, and 3 Surgery, The Research Institute of Clinical Medicine, Brain Korea 21 Program for Medical Science, Chonbuk National University Medical School and Hospital, Jeonju, Jeonbuk, South Korea and 4 Department of Pathology, Seoul National University College of Medicine, Chongno-gu, Seoul, South Korea

Requests for reprints: Dae-Ghon Kim, Division of Gastroenterology and Hepatology, Department of Internal Medicine, The Research Institute of Clinical Medicine, Chonbuk National University Medical School and Hospital, 634-18 Keumam-dong, Dukjin-ku, Jeonju, Jeonbuk 561-172, South Korea. Phone: 82-63-250-1681; Fax: 82-63-254-1694; E-mail: daeghon{at}chonbuk.ac.kr.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Cyclin B2, a G2-M cyclin, is overexpressed in colorectal adenocarcinomas compared with the normal mucosa. This study examined the level of cyclin B2 overexpression according to the histologic findings and investigated the mechanism(s) and clinical implications of cyclin B2 overexpression in colorectal adenocarcinomas.

Experimental Design: The immunoreactivity of the polyclonal antibodies to cyclin B2 was determined in colorectal cancer cells. The transcriptional regulation of cyclin B2 by NF-Y was analyzed using an in vitro transfection assay and an in vivo chromatin immunoprecipitation assay. The proliferative activity of the colorectal cancer cells in relation to cyclin B2 overexpression was further examined.

Results: The cytoplasmic distribution of cyclin B2 immunoreactivity was positive in 42 of 65 (64.6%) cases of colorectal adenocarcinoma, and the level was similar regardless of the histologic type. A dominant-negative form of NF-YA effectively inhibited the cyclin B2 promoter activity, and NF-Y was found to bind three conserved CCAAT boxes in the cyclin B2 promoter in colorectal adenocarcinoma cells. Tumor cells with a higher functional cyclin B2 activity grew faster than those with a lower activity. Furthermore, there was a correlation between the cells showing immunoreactivity to cyclin B2 and those containing the proliferating cell nuclear antigen, a G1-S cyclin, which is also downstream of NF-Y in colorectal adenocarcinoma cells.

Conclusions: Cyclin B2 seems to be a molecular marker of a colorectal adenocarcinoma and that its up-regulation and coordinate expression of the other cell cycle–related genes by NF-Y might contribute to tumor cell proliferation by accelerating cell cycle progression.

Mitosis is induced by the activation of the M-phase–promoting factor, which is a protein kinase whose principal subunits are p34cdc2 and cyclin B. Cyclin B is a regulatory subunit that varies in abundance throughout the cell cycle (1). The metaphase to anaphase transition marks the end of mitosis and is induced by the degradation of cyclin B, which in turn leads to the inactivation of M-phase–promoting factor. As the cell cycle progresses, cyclin B begins to accumulate when the cell enters the interphase, which is followed by the reactivation of M-phase–promoting factor and another round of mitosis. Therefore, the B-type cyclins are the primary mitotic cyclins, although several yeast B-type cyclins have been reported to function earlier in the cell cycle, particularly in the S phase (2, 3). There are two mammalian B-type cyclins, cyclins B1 and B2, which differ in their NH2 termini but have 57% similarity (4–6). Cyclins B1 and B2 are coexpressed in most dividing cells and, in association with cdc2, play a role in entering the G2-M phase, although their subcellular localization differs (7). Cyclin B1 is primarily cytoplasmic but constantly shuttles between the nucleus and the cytoplasm during the interphase. At the end of the prophase, cyclin B1 rapidly translocates to the nucleus (8, 9) and binds to the mitotic apparatus (10, 11). In contrast, cyclin B2 is primarily located in the Golgi apparatus in both the interphase and mitosis (5, 12). Cyclin B1 is an essential gene and its deletion results in embryonic lethality. In contrast, cyclin B2 knockout mice develop normally but are smaller than normal mice and have reduced litter sizes, which suggest that cyclin B2 has some growth advantage (12). Because cyclins B1 and B2 are important for the G2-M transition, regulating their expression has implications in controlling the cell cycle checkpoint. The tumor suppressor gene p53 down-regulates the transcription of both cyclin B1 and cyclin B2 and exerts some control over the G2-M checkpoint (13).

An analysis of the promoters of the cell cycle regulatory genes showed the common structural features to be a low frequency of TATA boxes and the presence of GC and/or CCAAT boxes, which are recognized by SP1 and NF-Y, respectively (14). Elements of the proximal promoter generally have some control of the cell cycle, often by boxes in close proximity to the start site(s). However, distant enhancer elements generally have little influence. Several such promoters contain multiple NF-Y–binding CCAAT boxes close to the cap site. Among them, cyclin B1 has two such CCAAT boxes and CDC25C has three, which are essential for transcriptional regulation (15–17). Cyclin B2 is also strictly regulated at the transcriptional level in cycling cells. The mouse promoter of cyclin B2 was cloned and three conserved CCAAT boxes that bind to the NF-Y trimeric transcription factors were found (18). NF-Y is a heteromeric protein consisting of three subunits, NF-YA, NF-YB, and NF-YC, which are all essential for DNA binding (19). The mRNA and protein levels of the histone fold containing NF-YB and NF-YC are constant, whereas the NY-YA subunit, but not its mRNA, reaches a maximum in the mid-S phase and decreases in the G2-M phase. In addition, the electrophoretic mobility shift assay confirmed that the CCAAT-binding activity follows the amount of NF-YA, suggesting that the NF-YA subunit is limiting within the NF-Y complex. This suggests that the NF-YA levels are regulated by a posttranscriptional mechanism (18).

This study used a polyclonal antibody to human cyclin B2, which is reactive for the transient expression of human cyclin B2 in an in vitro cell culture system, and examined the expression of cyclin B2 in both nonneoplastic and neoplastic lesions in the colorectal epithelium. Interestingly, cyclin B2 expression was strongly associated with a colorectal adenocarcinoma but the level was similar regardless of the histopathologic types. Furthermore, the immunoreactivity of cyclin B2 was found to be consistent with those of the NF-YC subunit. Therefore, the transcriptional regulation of cyclin B2 by NF-Y in colorectal adenocarcinomas was examined using in vitro and in vivo cell systems.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Tissue samples and histopathology. Specimens of histologically normal or adenocarcinoma tissues from the colorectal epithelium were obtained from the Chonbuk National University Hospital (Jeonju, Jeonbuk, South Korea). The tissues derived from surgical resections were rapidly fixed in 10% buffered formalin and embedded in paraffin for the standard histopathology analyses. The patients were enrolled in this study according to their age, tumor site, histopathologic grade, tumor-node-metastasis stage, and survival (Table 1 ). This study was approved by the Research Ethics Committee of Chonbuk National University Hospital.


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  		Table 1. Human cyclin B2 expression in the colorectal adenocarcinoma samples according to the clinicopathologic characteristics

 
Northern blot analysis. The membranes and Northern blots of the human multiple tissue (Clontech, Palo Alto, CA) containing 2 µg of polyadenylated RNA were hybridized with a human cyclin B2 cDNA probe generated from digested cDNA insert labeled with [{alpha}-32P]dCTP (3,000 Ci/mmol; NEN Life Science Products, Boston, MA) by random priming as described earlier (20). Subsequently, the blots were stripped and reprobed with radiolabeled human 18S according to the manufacturer's instructions.

Cell culture, transfection, and immunofluorescence. Human colorectal cancer cells (HT29, NCI-H630, NCI-H508, SNU-C2A, and NCI-H498) were obtained from the American Type Culture Collection (Rockville, MD). The cells were cultured in DMEM supplemented with 10% fetal bovine serum in air containing 5% CO2. The cyclin B2 eukaryotic expression vector was constructed as follows: the full-length human cyclin B2 gene derived from pBluescript(SK–)-CCNB2 digested with the EcoRI and XbaI restriction enzymes was cloned into pEGFP-C2 (Clontech) at the EcoRI/XbaI restriction enzyme sites in-frame. The transfection of the cyclin B2 gene into the HT29 cells was done using Lipofectin (Life Technologies, Grand Island, NY) according to the manufacturer's protocol. The full-length murine or human NF-YA, NF-YB, and NF-YC genes (21) were cloned in-frame into pEGFP-C1 (Clontech), pDeRed2-C1 (Clontech), and pEGFP-C2, respectively. The cells were grown on glass coverslips, fixed with 4% paraformaldehyde, permeabilized in PBS containing 0.2% Triton X-100, and blocked with 1% bovine serum albumin. The cells were then incubated with the primary antibody overnight at 4°C, washed, and further incubated with tetramethylrhodamine isothiocyanate isomer R–conjugated swine anti-goat immunoglobulin for 1 h. After the final wash, the cells were stained with 1 µg/mL Hoechst 33258 for 15 min to visualize the nuclei and mounted with 50% glycerol in PBS at 4°C. The cells were examined by laser scanning microscopy (LCM510, Carl Zeiss, Jena, Germany).

Immunohistochemistry and immunoblotting. Formalin-fixed, paraffin-embedded specimens (including normal, well-differentiated, moderately differentiated, poorly differentiated, or mucinous colorectal adenocarcinoma) were sectioned at a thickness of 4 µm. Deparaffinization and rehydration were carried out using a graded series of xylene and alcohol, respectively. The sections were then treated with 0.3% hydrogen peroxide for 3 min and a blocking antibody for 30 min. The sections were incubated for 1 h with the primary antibody, goat polyclonal cyclin B2 (N-20), rabbit polyclonal NF-YA (CBF-B, H-209), rabbit polyclonal NF-YB (CBF-A, FL-207), or goat polyclonal NF-YC antibody (CBF-C, H120), all of which were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and diluted 1:100 in 1 mol/L Tris buffer (pH 8.0). Detection was done using the avidin-biotin-peroxidase complex method (DAKO, Glostrup, Denmark), and 3-amino-9-ethylcarbazole was used as the chromogen. Counterstaining was done using Meyer's hematoxylin. The percentage of positive cells (>10%) and the staining intensity (more than weak) of the tumors were evaluated. The samples incubated with PBS or mouse IgG1 instead of the primary antibody were used as the negative controls. For immunoblotting, the primary colorectal cancer and surrounding nontumor tissues were obtained from the surgical resections of a colorectal cancer patient who provided written informed consent. The tissues were lysed in 1% Triton X-100 in the presence of aprotinin (1 unit/mL trypsin), leupeptin (10 µg/mL), and pepstatin A (10 µg/mL). The extracted proteins were resolved by SDS-PAGE and transferred to a nitrocellulose membrane. The membranes were incubated with the primary cyclin B2 antibody (at 1:100 dilution) for 1 h at room temperature. After incubation, the blots were washed thrice in PBS/0.1% Tween 20. Immunoreactive cyclin B2 was detected with alkaline phosphatase–conjugated goat anti-rabbit IgG using a commercial chemiluminescence detection kit (Amersham Biosciences, Uppsala, Sweden) according to the manufacturer's instructions.

Luciferase assay. The human cyclin B2 promoter-driven luciferase reporter construct B2H-Luci was a kind gift from Dr. Kurt England (University of Leipzig, Leipzig, Germany; ref. 22). The murine cyclin B2 promoter-driven luciferase reporter construct B2-Luci and the CCAAT mutants Y1,2m and Y1,2,3m were obtained from Dr. Roberto Mantovani (Institut de Chimie Biologique, Strasbourg, France; ref. 18). The NF-YA expression vector and the dominant-negative NF-Y vector were originally obtained from Dr. Roberto Mantovani (21) and cloned into pcDNA3.1(–) of the EcoRI and BamHI sites in the sense orientation. For the cotransfection experiments, the cells were cotransfected with the luciferase reporter constructs and the mutant expression vector NF-YA using Lipofectin. The control experiments were carried out using pGL3Basic (Promega, Madison, WI). The cells were plated in 24-well plates at 2 x 104 per well. Eighteen hours later, the cells were incubated with 500 ng of the B2H-Luci plasmid for 16 h at 37°C. After transfection, the cells were replenished with complete medium and incubated for 36 h. The cells were then lysed in 120 µL of a reporter lysis buffer (Promega) and stored at –20°C until assayed. The activity of the experimental reporter was normalized to the activity of the internal control to minimize the experimental variability caused by differences in the cell viability and/or transfection efficiency. The luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions. The cells were cotransfected with pRL-TK (Promega) and harvested. The firefly and Renilla luciferase activities were measured for normalization using a luminometer (Lumat LB9507, Berthold, Bad Wildbad, Germany).

Chromatin immunoprecipitation. For cross-linking, formaldehyde was added directly to the cell culture medium of the normal growing colorectal carcinoma cells at a final concentration of 1%. Fixation proceeded at room temperature for 10 min and was quenched by adding glycine to a final concentration of 0.125 mol/L. The cells were rinsed twice with cold PBS, including the protease inhibitor. The cells were then scraped from the plates, collected by centrifugation, and swelled in 10 mmol/L HEPES/KOH (pH 7.9), 1.5 MgCl2, and 10 mmol/L KCl, including a protease inhibitor. After 10 min of incubation on ice, the cells were vortexed for 10 s and the nuclei were collected by centrifugation. The nuclei were resuspended in a lysis buffer [50 mmol/L Tris-HCl (pH 8.1), 10 mmol/L EDTA, 1% SDS, protease inhibitors] and incubated on ice for 10 min. A chromatin solution was sonicated five times for 10 s with a 10-s cooling period between each pulse using a Labsonic sonicator (Braun, Melsungen, Germany). The samples were centrifuged at 20,000 x g for 5 min. A part of the supernatant was retained as the total chromatin input and processed with the eluted immunoprecipitates beginning at the cross-link reversal step as described previously. The rabbit polyclonal anti-NF-YA antibody (H-209; 1 µg) was added to the precleared chromatin solution and then rotated overnight at 4°C. A mixture of 0.5 µg of the rabbit polyclonal anti–cyclin B1 antibody (H-433) and 0.5 µg of the goat polyclonal anti–cyclin B2 antibody (N-20) was used as the negative control. The immunocomplex was eluted by resuspending protein A-Sepharose (Amersham Biosciences) in an elution buffer and incubated at room temperature for 45 min. The samples were microfuged and the elution step was repeated. The eluates were combined and NaCl was added to a final concentration of 0.3 mol/L. The cross-links were reversed by incubating the samples at 67°C for 5 h followed by precipitation with 2 volumes of ethanol overnight at –20°C and microcentrifugation. The pellet was dissolved in 100 µL TE [10 mmol/L Tris (pH 8.0), 1 mmol/L EDTA] and 25 µL of 5x proteinase K buffer [1.25% SDS, 50 mmol/L Tris (pH 7.5), 25 mmol/L EDTA] and 1.5 µL proteinase K (10 mg/mL; Sigma, St. Louis, MO) and incubated at 45°C for 1 h. The DNA was extracted with phenol/chloroform and precipitated with ethanol. PCR was done using Taq DNA polymerase (Promega) according to the manufacturer's protocol with the following primers (23): cyclin B2, 5'-CCAGAAGAGGAATAAAGGCCAACCAACTTCCG-3' and5'-GGGCGTCGGAGCAGCGCCATGGGGGACGGGG-3'; cyclin B1, 5'-GCGCAGGCGCAGAGGCAGACCACGTGAGAG-3' and 5'-TTCACCAGGCAGCAGCTCAGCGGGGAGAAG-3'; and ANXA8, 5'-AAGGTGGCTGTGCCGCCTGCCACCTTCTCA-3' and 5'-CTCTTTCACCTCGGGGGCACCTTTCCCAGG-3'. The PCR products were analyzed on 2% agarose gels and stained with ethidium bromide.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test. The viable cells were adjusted to a concentration of 1 x 104/mL with the medium and plated in 24-well plates. The cells were incubated for 5 days and the level of cell proliferation was examined by measuring the cell viability using the tetrazolium salt test described elsewhere (20). Briefly, 50 µL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (5 mg in 0.9% NaCl; Sigma) were added and the mixture was incubated for 4 h at 37°C. The precipitated dye was dissolved in 200 µL DMSO (Sigma) and in a 50 µL glycine buffer (pH 10.5), and the absorbance was read at a wavelength of 540 nm using an ELISA reader (SpectraMax 340, Molecular Devices).

Scoring and statistical analysis. The specimens were regarded as being positively stained if >10% of the epithelial cells showed a reaction at a low magnification. A {chi}2 test or two-tailed Fisher's exact test was used to compare the staining results according to the independent groups at a significance level of 5%. All the data were entered into Microsoft Excel 5.0, and paired t tests were done using GraphPad software. P values of <0.05 were considered significant. We created Kaplan-Meier survival curves using Statistical Package for the Social Sciences 13.0 and used the Mantel-Cox log-rank test to calculate the statistical significance (P value) of difference between survival curves.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Cyclin B2 expression in gastrointestinal tissues and colorectal adenocarcinoma. Previously, a cDNA clone, as an expressed sequence tag, was isolated from a human fetal liver cDNA library and identified as a murine cyclin B2 homologue. The sequence of human cyclin B2 is registered in the Genbank sequence database (accession no. AF002822). The level of cyclin B2 mRNA expression was examined in gastrointestinal tissues because they contain various proliferating, secretory, and absorptive functional cells. The gastrointestinal tissues expressed cyclin B2 mRNA, with the highest level being observed in the colon followed in order by the small intestine and stomach (Fig. 1A ). Cyclin B2 expression in the colorectal adenocarcinoma and corresponding nontumor tissues was examined to determine if there is any difference in the cyclin B2 expression level between colorectal adenocarcinomas and normal tissue. The cyclin B2 protein was preferentially overexpressed in the tumor tissues. Six of nine (66.7%) colorectal adenocarcinomas were tested positive (Fig. 1B). The specific immunoreactivity of cyclin B2 by goat polyclonal cyclin B2 was determined by transfecting the 293T cells with Myc-tagged cyclin B2 expression vector and assaying the cyclin B2 antibody reactivity by immunoblot analysis. The transfected cyclin B2 cDNA contained a Myc tag in the COOH terminus. Therefore, an immunoblot on the same membrane was also done with an anti-c-Myc mouse monoclonal antibody (9E10, Santa Cruz Biotechnology). The cyclin B2 antibody specifically detected the band corresponding to the cyclin B2 protein (Fig. 1C).


Figure 1
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  		Fig. 1. Tissue expression of cyclin B2. A, human adult tissue poly(A)+ Northern blots were probed with 32P-labeled cyclin B2 (CCNB2) cDNA. B, differential expression of cyclin B2 in colorectal adenocarcinomas and nontumor tissues. The extracted proteins (40 µg)were resolved by SDS-PAGE and transferred to a nitrocellulose membrane. Bottom, the blot was incubated with the primary cyclin B2 antibody and then with the actin antibody as a loading control. C, protein lysates were prepared from 293T cells transfected with the Myc vector alone (lane 1) or Myc-tagged cyclin B2 (lane 2). The membranes were incubated with either anti-Myc or anti–cyclin B2 antibodies. Arrow, cyclin B2 protein. Numbers on the left, molecular weight markers.

 
Cyclin B2 expression in colorectal adenocarcinoma cells. HT29 cells transiently expressing green fluorescent protein (GFP)-cyclin B2 fusion protein were generated. The green fluorescence was observed in the cytoplasm of the cells, which merged perfectly with the immunofluorescence staining for cyclin B2. This ectopic expression of cyclin B2 showed a stronger immunoreactivity than the surrounding nontransfected cells, which suggests that cyclin B2 is mainly located in the cytoplasm of colorectal cancer cells (Fig. 2A ). Immunofluorescence staining was done in human colorectal adenocarcinoma cells (HT29) to determine the level of immunoreactivity of the polyclonal antibody (N-20) against endogenous cyclin B2. The immunoreactivity for cyclin B2 was observed in the cytoplasm of HT29 cells (Fig. 2B). Another colorectal adenocarcinoma cell line (NCI-H630) was used to confirm the cell cycle–dependent regulation of endogenous cyclin B2 expression detected using this antibody. Immunofluorescence staining showed that cyclin B2 accumulates in the prophase and metaphase in mitosis (G2-M) and then disappears during the metaphase to anaphase transition (Fig. 2C), which is consistent with previous reports (6, 23). This suggests that the polyclonal antibody used in this study effectively recognized the endogenous and ectopic expression of cyclin B2.


Figure 2
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  		Fig. 2. Cytoplasmic expression of the cyclin B2 protein. A, the cells (HT29) were transfected with 1 µg of the GFP-fused cyclin B2 expression vector or an empty vector pEGFP-C2. The cells were processed for 1 µg/mL Hoechst 33258 staining to visualize the nuclei (blue) and for indirect immunofluorescence staining of cyclin B2 (red). The cells were stained with 1 µg/mL Hoechst 33258 to visualize the nuclei (blue). The cell morphology was observed using laser scanning microscopy (LCM510). Trans, transmission. Bar, 10 µm. B, endogenous cyclin B2 stained with anti–cyclin B2 (red). The HT29 cells were stained with 1 µg/mL Hoechst 33258 to visualize the nuclei (blue). Bar, 10 µm. C, indirect immunofluorescence microscopy of human colorectal adenocarcinoma cells (NCI-H630). Endogenous cyclin B2 expression at various stages of mitosis. P, prophase; M, metaphase; A, anaphase; T, telophase. Bar, 10 µm.

 
Cyclin B2 immunoreactivity in adenocarcinoma of the colorectal mucosa. Cyclin B2 expression in the colorectal epithelium was examined using this polyclonal cyclin B2 antibody (N-20). Interestingly, no cyclin B2 immunoreactivity was detected in the normal mucosa of the colorectum (Fig. 3A ). The immunohistochemical examination was extended to carcinomas of the colorectum. The cytoplasmic distribution of cyclin B2 immunoreactivity was confined mainly to the epithelial cells in glandular carcinoma cells (Fig. 3B), whereas there was no cyclin B2 expression in the stromal and infiltrative blood cells. However, the germinal centers, which mainly contained B lymphocytes in the mucosal lymph follicles and lymph nodes in the nontumor tissues, were also tested positive for cyclin B2 immunoreactivity. There did not seem to be any correlation between the intensity of immunoreactivity and the level of tumor cell differentiation. Mucinous adenocarcinoma cells also showed strong immunoreactivity. Therefore, the frequency of cyclin B2 expression was examined according to the histopathologic classification of colorectal adenocarcinoma (Table 1). Nine of 14 (64.3%) well-differentiated adenocarcinomas (Fig. 3B), 21 of 31 (67.7%) moderately differentiated adenocarcinomas (Fig. 3C), 6 of 11 (54.5%) poorly differentiated adenocarcinomas (Fig. 3D), and 6 of 9 (66.7%) mucinous adenocarcinomas were tested positive (Fig. 3E). Compared with the undivided adenocarcinomas, there was no difference in the frequency of positive cyclin B2 immunoreactivity according to the histopathologic differentiation of colorectal adenocarcinomas. There was no significant difference in the frequency of positive cyclin B2 immunoreactivity according to the tumor stage. However, Kaplan-Meier survival analysis revealed positive cyclin B2 expression in colorectal adenocarcinomas to correlate with a better survival than negative cyclin B2 expression (Fig. 3F). This suggests that the cyclin B2 expression is more involved in colorectal oncogenesis than in tumor invasion and metastasis.


Figure 3
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  		Fig. 3. Immunohistochemical detection of cyclin B2 in the normal colorectal mucosa and colorectal adenocarcinoma. A, no cyclin B2–positive cells were detected in the normal colorectal epithelium. Original magnification, x40. B, cyclin B2 staining in a well-differentiated adenocarcinoma was localized mainly in the cytoplasm of the epithelial cells. The immunoreactivity had a stippled or spotted pattern and was barely detectable in the adjacent lamina propria, muscularis mucosa, submucosal vessels, or fibrous tissue. Original magnification, x100. C, cyclin B2–positive cells throughout the moderately differentiated adenocarcinoma epithelium. Original magnification, x100. D, staining for cyclin B2 was detected in the poorly differentiated adenocarcinoma associated with considerable stromal fibrosis of the colorectum. Original magnification, x200. E, staining for cyclin B2 was detected in the mucinous adenocarcinoma but not in the mucin pool and surrounding stromal cells. Original magnification, x200. F, Kaplan-Meier plot of the overall survival of patients with colorectal adenocarcinoma grouped based on cyclin B2 expression. The difference between groups was significant (P = 0.050, Mantel-Cox log-rank test).

 
Transcriptional regulation of cyclin B2 by NF-Y. Human and mouse cyclin B2 promoters contain three conserved CCAAT boxes, which have been shown to bind the NF-Y transcriptional factor (18). NF-Y consists of three subunits, NF-YA, NF-YB, and NF-YC, all of which are essential for DNA binding. NF-YB and NF-YC dimerization is vital for the NF-YA association and subsequent DNA binding. The effect of NF-Y and the three CCAAT boxes on the transcriptional activation in colorectal adenocarcinoma cells was determined. To this end, the YA13m29 dominant mutant of the NF-Y subunit A was used (22). The B2H-Luci reporter plasmid was cotransfected with a construct expressing YA13m29, and its level was assayed in the colorectal cancer cells. The promoter activity of cyclin B2 in colorectal cancer cells according to the level of tumor cell differentiation was determined by selecting colorectal cancer cells at different levels of differentiation, such as NCI-H630 (well differentiated), NCI-H508 (moderately differentiated), SNU-C2A (poorly differentiated), and NCI-H498 (mucinous differentiation). Their tumorigenicity at different levels of differentiation was examined using a xenograft model as described previously (24). The levels of cyclin B2 promoter activity seemed to be cell specific (i.e., higher in the colorectal cancer cells with well and moderate differentiation than in those cells with poor and mucinous differentiation). Dominant-negative NF-YA clearly reduced the activity of the human cyclin B2 promoter in all colorectal cancer cells by up to 16.4% to 52.6% (Fig. 4A ). Therefore, experiments were carried out to determine if there is a correlation between the reduction of cyclin B2 promoter activity and tumor cell proliferation. NCI-H508 cells, which showed the largest decrease in the cyclin B2 promoter activity by the dominant-negative NF-YA, grew faster than the other three tumor cell lines (Fig. 4B). This indicates a correlation between a higher functional cyclin B2 promoter activity and a higher proliferative activity.


Figure 4
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  		Fig. 4. Transcriptional regulation of cyclin B2 by NF-Y. A, inhibition of cyclin B2 transcription by a NF-YA dominant-negative vector. The colorectal carcinoma cells were transfected with the promoters from cyclin B2 driving luciferase transcription and 3 µg of the mutant-type NF-YA expression plasmids (YA13m29). The assay was done 24 h after transfection. Columns, mean of quadruplicate cultures from three independent experiments; bars, SE. *, P < 0.05. B, cell growth of the four types of colorectal cells. At the indicated time intervals, the monolayer cells were processed for the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and the absorbance was read at a wavelength of 540 nm using an ELISA reader. Points, mean of quadruplicate cultures from two independent experiments; bars, SE. *, P < 0.05. C, chromatin immunoprecipitation assays showing binding of NF-Y subunits to the cyclin B2 promoter. M, marker. Lane 1, NF-YA subunit; lane 2, NF-YA + NF-YB + NF-YC subunits; lane 3, control; lane 4, input. D, a mutation of the CCAAT boxes impairs the cyclin B2 promoter activity. The B2-Luci vector (500 ng) carrying the wild-type murine cyclin B2 promoter, the Y1,2m vector carrying a mutation in two CCAAT boxes, or the Y1,2,3m vector carrying a mutation in all three CCAAT boxes was transiently cotransfected in the four types of colorectal cells. Columns, mean of quadruplicate cultures from three independent experiments; bars, SE.

 
Previously, NF-Y protein binding in vitro to the identical CCAAT boxes in the mouse cyclin B2 promoter was shown by electrophoretic mobility shift assay (18). Therefore, a chromatin immunoprecipitation assay was carried out to test for in vivo protein binding in colorectal cancer cells. Using the DNA fragment carrying the three conserved CCAAT boxes in chromatin immunoprecipitations yielded a signal for NF-Y on the cyclin B2 promoter (Fig. 4C). The cyclin B1 gene was used as a positive control for binding by NF-Y (25). The ANXA8 promoter served as the negative control for NF-Y binding. The results suggest that NF-Y was the major activating transcription factor on the cyclin B2 promoters acting through the three conserved CCAAT boxes. The functional contribution of the three CCAAT boxes to the up-regulation of the promoter activity in colorectal cancer cells was next examined. Constructs with a CCAAT to TTACT mutation in the cyclin B2 promoter [two luciferase constructs carrying two mutated elements (Y1,2m) or all three mutated CCAAT boxes (Y1,2,3m)] were used for this purpose (18). The cyclin B2 promoter activity was almost abolished by the mutations in all or in only two of the CCAAT boxes. However, mutations in only two CCAAT boxes effectively impaired the promoter activity in all cancer cells to <10% (Fig. 4D). This suggests that the cyclin B2 promoter activity is mainly modulated by the CCAAT boxes 1 and 2 of the cyclin B2 promoter.

NF-Y immunoreactivity in colorectal adenocarcinoma. The specific immunoreactivity of the three NF-Y subunits via the primary antibody against NF-YA, NF-YB, and NF-YC was determined by transfecting the NCI-H508 cells with either the GFP-fused NF-YA, red fluorescent protein–fused NF-YB, or GFP-fused NF-YC expression vector, respectively. A comparison of the immunofluorescence and transmission images revealed the dominant nuclear localization to be in those cells expressing the ectopic GFP-fused NF-YA. The green fluorescence perfectly overlapped with the NF-YA immunoreactivity. In contrast, cytoplasmic and/or nuclear localization of the ectopic GFP-fused NF-YB or NF-YC was detected, and the green or red fluorescence was overlapped perfectly with the NF-YB or NF-YC immunoreactivity, respectively (Fig. 5A ). The same polyclonal antibodies against NF-YA, NF-YB, and NF-YC were used to determine the subcellular localization of the expression of the three endogenous NF-Y subunits in the colorectal adenocarcinoma cells. NF-YA and NF-YB immunoreactivity was always confined to the nucleus of these cells. In contrast, NF-YC immunoreactivity was localized in the cytoplasm and/or nuclei of the tumor cells. Cyclin B2 was located exclusively in the cytoplasm of the tumor cells as described earlier (Fig. 5B). The subcellular localization of NF-YC became nuclear when GFP-fused NF-YC was coexpressed with red fluorescent protein–fused NF-YB (Fig. 5C), which suggests that the nuclear localization of NF-YC also depends on the presence of its histone fold partner, NF-YB, in colorectal tumor cells as reported elsewhere (26).


Figure 5
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  		Fig. 5. Immunofluorescence assay of the NF-Y subunits in colorectal adenocarcinoma cells. A, NCI-H508 cells were transfected with 1 µg of the expression vector of the GFP-fused NF-YA, red fluorescent protein (RFP)–fused NF-YB, GFP-fused NF-YC, or an empty vector pEGFP-C2. The cells were processed for indirect immunofluorescence (IF) staining for NF-YA (red), NF-YB (green), and NF-YC (red). The cells were stained with 1 µg/mL Hoechst 33258 to visualize the nuclei (blue). Bar, 10 µm. B, endogenous NF-Y subunits stained with anti-NF-YA (red), anti-NF-YB (green), and anti-NF-YC (red). NCI-H508 cells were stained with 1 µg/mL Hoechst 33258 to visualize the nuclei (blue). Bar, 10 µm. C, the NCI-H508 cells were cotransfected with red fluorescent protein–fused NF-YB expression vector and GFP-fused NF-YC expression vector together. Bar, 10 µm.

 
The expression level of the three NF-Y subunits in the paraffin-embedded tissue section obtained from samples of the colorectal adenocarcinoma and surrounding nonmalignant tissue was next examined. Positive staining for NF-YA or NF-YB was observed in the nucleus and cytoplasm of the malignant colorectal epithelium and the normal epithelium with proliferative activity at the crypt base (Fig. 6B, C, F, and G ). In contrast, immunoreactivity for NF-YC was localized to the cytoplasm of the malignant colorectal epithelium but not in the cytoplasm of the normal epithelium at the crypt base, which is similar to the results observed for cyclin B2 immunoreactivity (Fig. 6A, D, E, and H). This suggests that NF-YC and cyclin B2 expression is related to the proliferation of tumor cells, whereas NF-YA and NF-YB expression is related to the proliferation of tumor cells and the normal epithelium in colorectal cancers.


Figure 6
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  		Fig. 6. Immunohistochemical reactivity of NF-Y subunits in colorectal adenocarcinoma. A and E, cytoplasmic immunoreactivity for cyclin B2 was positive in the adenocarcinoma epithelium but negative in the normal epithelium at the crypt base. B and F, positive nuclear and cytoplasmic immunoreactivity for NF-YA can be seen in the adenocarcinoma epithelium as well as in the normal epithelium at the crypt base. C and G, positive nuclear and cytoplasmic immunoreactivity for NF-YA can be seen in the adenocarcinoma epithelium as well as in the normal epithelium at the crypt base. D and H, cytoplasmic immunoreactivity for NF-YC was positive in the adenocarcinoma epithelium but negative in the normal epithelium at the crypt base.

 
Coexpression of proliferating cell nuclear antigen in colorectal adenocarcinomas. Proliferating cell nuclear antigen (PCNA) is an auxiliary factor necessary for DNA polymerase activity, which is essential for both DNA replication and repair (27). PCNA is expressed in cycling cells and is used frequently to measure the proliferative activity of tissues (28). In addition, it is the most frequently observed protein among cell cycle–associated proteins (29). The PCNA promoter contains two CCAAT boxes at nucleotides –95 and –145 (30). Therefore, this study examined the level of PCNA expression in colorectal cancer tissue as well as in the mucosal lymph follicles and lymph nodes in nontumor tissues. Interestingly, most cyclin B2–positive colorectal cancer tissues showed positive nuclear immunoreactivity for PCNA (Fig. 7A and B ). In addition, the germinal centers in the mucosal lymph follicles and lymph nodes were also tested positive for both PCNA and cyclin B2 (Fig. 7C-F). The effect of NF-Y on the PCNA promoter activity in colorectal adenocarcinoma cells was then determined. The pGL3-pnca reporter plasmid (31) was cotransfected with a construct expressing YA13m29 and assayed in colorectal cancer cells according to the tumor histopathology. Dominant-negative NF-YA clearly reduces the activity of the human PCNA promoter in NCI-H508 cells, which are the most rapidly growing cells, to 6.3% (Fig. 7G). This suggests that NF-Y increases the functional activity of PCNA and cyclin B2 to a higher level in NCI-H508 cells than in other cells. Considering that PCNA is a G1-S cyclin, its higher expression level seems to be linked to the aberrant expression of cyclin B2, a G2-M cyclin, and to the tumor cell cycle progression in colorectal adenocarcinomas.


Figure 7
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  		Fig. 7. Coexpression of cyclin B2 and PCNA. A and B, positive immunoreactivity for both cyclin B2 and PCNA can be seen in the paraffin-embedded tissue sections of colorectal adenocarcinoma. C and D, cyclin B2–immunostained and PCNA-immunostained positive lymphoid cells in the germinal center of the mucosal lymph follicles in the nontumor tissues. E and F, cyclin B2–immunostained and PCNA-immunostained positive lymphoid cells in the germinal centers of the lymph node in nontumor tissues. G, inhibition of PCNA transcription by a NF-YA dominant-negative vector. The colorectal carcinoma cells were transfected with the promoters from PCNA driving luciferase transcription and 3 µg of mutant-type NF-YA expression plasmids (YA13m29). The assay was done 24 h after transfection. Columns, mean of quadruplicate cultures from three independent experiments; bars, SE. *, P < 0.05.

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
In vertebrate cells, cyclin B1 and B2 levels increase during the G2 phase and peak during mitosis. In the interphase, cyclin B1 is mainly located in the microtubules, whereas cyclin B2 is found in the Golgi apparatus (5). Many studies have reported the accumulation of cyclins B1 and B2 in malignant tumors, such as colorectal and lung cancer (32–34). However, the molecular mechanism underlying this deregulation has not been elucidated. The overexpression of cyclins B1 and B2 contributes to the chromosomal instability observed in cancer cells through an alteration of the spindle checkpoint (33). The accumulation of the cyclin B1 protein might indicate a defect in a post-translational deregulation in the absence of mRNA overexpression or point mutations. In contrast, a subset of malignant tumors shows cyclin B2 mRNA expression, and its overexpression is not due to gene amplification (33). Most colorectal cancers express high levels of cyclin B1, which is consistent with the high rate of cell proliferation (32). High levels of cyclin B1 expression are a frequent and early event in colorectal carcinomas. This study found that cyclin B2 is also overexpressed in colorectal adenocarcinomas and that its expression level is similar regardless of the histopathologic status. Furthermore, the expression of the NF-Y subunits was immunohistochemically confirmed in colorectal adenocarcinomas using a set of polyclonal antibodies against the endogenous and ectopic NF-Y subunits.

Three CCAAT boxes are conserved in the human and mouse cyclin B2 promoter. Both their nucleotide sequences and the 33-bp distance from each other are identical (23). These elements bind the trimeric transcriptional activator NF-Y (17, 19). Binding and activation through NF-Y dominates the general transcriptional activity from the human cyclin B2 promoter. To determine if NF-Y is responsible for the cyclin B2 transcriptional activation in colorectal cancer cells, the YA13m29 dominant-negative mutant of NF-Y subunit A was first used, and mutations in all three or only two of the CCAAT boxes were assayed in the cells with a different histopathologic status. In other experiments, chromatin immunoprecipitations were done to test for the in vivo protein binding of the NF-Y subunits to the cyclin B2 promoter. Both experiments indicated NF-Y to be the major activating transcription factor on the cyclin B2 promoters acting through the three conserved CCAAT boxes regardless of histopathologic status. However, the functional cyclin B2 promoter activity, which is inhibited by YA13m29, is proportional to the level of cell proliferation. The oncogenic cooperation of the two major regulators of the cell cycle, mutant p53 and NF-Y, is essential for the binding of p300. This complex overexpresses the cell cycle genes, which allows the cells in colorectal cancers pretreated with adjuvant chemotherapy to escape from cell proliferation control (35). The NCI-H630 and NCI-H508 cells were derived from patients who had previously undergone chemotherapy (24). The genes activated in the G2-M phase (CDC25C and cyclin B1/cyclin B2) and other genes activated in the G0 (PDGFß-R), G1-S (E2F1, CDC25A, and PCNA), S (cyclin A and CDC25B), and G2-S phases (topoisomerase II{alpha}and PLK) are controlled by NF-Y, and their promoters contain CCAAT boxes, although it is unclear how NF-Y distinguishes between the different targets in vivo in any given phase of the cell cycle (17, 36–42). Therefore, in addition to cyclin B2 expression, the expression of cyclin A, cyclin B1, cyclin-dependent kinase 1, and cdc25C was also much higher in the colorectal cancer cells and tissue samples, which is mediated by the mutant p53 and NF-Y protein complex (35).

The NF-Y subunits evidently bind to the DNA fragment of the cyclin B2 promoter and regulate its promoter activity in adenocarcinoma cells. The binding of NF-Y to the cell cycle that regulates the promoters during the different phases of the cell cycle in vivo was assayed using chromatin immunoprecipitation analysis (43). NF-YB and NF-YC dimerization are essential for the NF-YA association and sequence-specific DNA binding. NF-YB and NF-YC contain the histone fold motif found in all core histones, which mediate dimerization and DNA binding (44). Recently, NF-YA was reported to contain a nonclassic nuclear localization signal at its COOH terminus (amino acids 262-317) and be translocated to the nucleus by the importin ß–mediated pathway. In addition, it was reported that the NF-YB/NF-YC heterodimer is translocated into the nucleus in an importin 13–dependent manner (45). The results in the present study also showed that ectopic and endogenous NF-YA is always nuclear at the steady state, which suggests that NF-YA is imported rapidly into the nucleus independently of NF-YB and NF-YC. In contrast to the dominant cytoplasmic localization of the ectopic and endogenous NF-YC, the ectopic NF-YB is transported to both the cytoplasm and the nucleus. However, endogenous NF-YB is imported exclusively to the nucleus. Therefore, NF-YB and NF-YC are slowly imported into the nucleus. Considering that a low level of ectopic NF-YB expression is easily imported into the nucleus (data not shown), it can be seen that a physiologic process will be needed for the nuclear translocation of NF-YB. In contrast, the nuclear translocation of NF-YC, which is dependent on the coexpression of NF-YB, seemed to be cell cycle specific as described previously (26). The NF-YC changes and their accumulation during the cell cycle, which are cytoplasmic during the G0 and G1 phases, were located in the nucleus at the onset of the S phase. Therefore, the nuclear localization of NF-YC is closely related to DNA synthesis and proliferation in colorectal cancers. The different kinetics of nuclear translocation between the histone fold partners NF-YB and NF-YC will require further study. Immunohistochemical analysis of the expression of the NF-Y subunit in colorectal adenocarcinoma tissues revealed the dominant nuclear localization of NF-YA and NF-YB and the dominant cytoplasmic localization of NF-YC.

Human MHC class II genes are activated at the mature B-cell stage. However, progression to the plasma B-cell stage after being stimulated with foreign antigens, various mitogens, and T-cell factors results in the uniform repression of the expression of all class II genes at the transcriptional level (46). NF-Y is associated with the protein cofactor PC4, which might play an important role in the NF-Y–mediated transcriptional control of the class II genes in B lymphocytes (47). In addition to the adenocarcinoma tissues, cyclin B2 was also found in the germinal centers containing mainly B lymphocytes in the mucosal lymph follicles and lymph node in nontumor tissues. Therefore, NF-Y–mediated cyclin B2 expression might play a role in B lymphocyte maturation in the germinal centers. These results on PCNA expression are consistent with those from a previous study in that the germinal center in mucosa-associated lymphoid tissue shows proliferative activity and PCNA expression (48). Therefore, the expression of cyclin B2 in concert with other cell cycle–regulated genes seems to be modulated by NY-YB in the germinal center of mucosa-associated lymphoid tissue as well as in colorectal cancer.

In summary, cyclin B2 overexpression was observed in colorectal adenocarcinomas with a similar level regardless of the histopathologic status. Furthermore, the NF-Y subunits evidently bind to the DNA fragment of the cyclin B2 promoter and regulate its promoter activity in adenocarcinoma cells. Cyclin B2 up-regulation and the coordinate expression of the other cell cycle–related genes by NF-Y might contribute to tumor cell proliferation by accelerating cell cycle progression.


   Acknowledgments
 
We thank Drs. Kurt England, Roberto Mantovani, Benjamin Y.M. Yung, and Deug Y. Shin for providing plasmids, the Korea Basic Science Institute for the laser scanning microscopy, and all the patients who participated in the clinical studies that made this work possible.


   Footnotes
 
Grant support: 21C Frontier Human Genome Project grant FG-1-2 and Korea Research Foundation grants F00351 and F00153.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 6/19/06; revised 11/ 5/06; accepted 11/17/06.


   References
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 Materials and Methods
 Results
 Discussion
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