The human embryonic kidney cell line 293 purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and the human hepatocellular carcinoma cell line HepG2 were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Wako Pure Chemical Industries, Ltd., Osaka, Japan) supplemented with 10% fetal bovine serum (FBS; Life Technologies, Inc., Rockville, MD, USA) in a humidified incubator with 5% CO₂ at 37°C. The murine mammary tumor cell line Cl66M2 (M2) was generously provided by Dr Rakesh K. Singh (University of Nebraska Medical Center, Omaha, NE, USA) (18) and cultured in DMEM supplemented with 5% FBS in a humidified incubator with 5% CO₂ at 37°C.

The TRB3 flag-tagged expression vector was constructed by ligating the full length human TRB3 cDNA into BamHI and XhoI restriction sites of pcDNA3.1-Hygro (Life Technologies, Inc.) (9). The construct was verified by sequencing.

The expression vector pcDNA3.1-Hygro-flag-human TRB3 was transfected into Cl66M2 cells using Lipofectamine 2000 reagent (Life Technologies, Inc.). This cell line was termed M2TRB3. After transfection, the clone of the cells stably expressing M2TRB3 was selected by a limiting-dilution method in culture media supplemented with hygromycin. The cells transfected with the empty vector were also prepared as the control (M2mock). In M2TRB3 and M2mock cells, the levels of mRNA and protein expression were confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and western blot assays.

These assays were performed as described previously by us (15). Two murine mammary tumor cell lines M2TRB3 and M2mock were plated into 6-well 35-mm diameter culture plates (1.0×10⁴ cells/well) in DMEM containing 10% FBS. Cells were starved in DMEM containing 0.5% FBS for 48 h. After starvation, the culture media were removed and cells were grown in DMEM containing 10% FBS for the indicated time course (0–72 h). The cells were washed twice with phosphate-buffered saline (PBS), harvested, resuspended in 1 ml PBS and the number of cells was determined using a hemocytometer Burker-Turk (Erma Inc., Tokyo, Japan). Each assay was repeated more than three times to confirm the results. The number of cells was plotted on a time-response curve as indicated in the figures.

Male four-week-old BALB/cSlc-nu/nu mice obtained from Japan SLC, Inc. (Shizuoka Japan) were used. M2, M2TRB3 and M2mock cells (1.0×10⁶/200 μl) were subcutaneously inoculated into the right lower flank of the mice. Tumor diameters (mm) and body weight (g) were recorded twice weekly. The tumor volume (mm³) was calculated by the formula: Volume = L × D × H × π/6, where L is the length, D is the depth, and H is the height. At 35 days after inoculation, all mice were euthanized and complete autopsies were performed. Animal experiments were conducted in accordance to the regulations specified by the Institutional Animal Use and Care Committee of Nagoya City University.

These assays were performed using an established method as described previously by us (16). Paraffin sections (3-μm) were prepared to include tumors resected from the lower flank of each mouse. These sections were treated in 3% H₂O₂ for 10 min to block the endogenous peroxidase activity. For antigen retrieval, the sections were brought to boiling in 0.1 M citrate buffer, pH 6.0. Sections were incubated with a primary antibody of PCNA (1:500 dilution) (sc-56; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) at room temperature for 60 min. After incubation with the secondary antibody, sections were then stained using an ABC kit (Vector Laboratories, Inc., Burlingame, CA, USA) according to the manufacturer’s instructions. The PCNA labeling index was determined by calculating the ratio of PCNA-positive nuclei/total number of nuclei counted. Ten high power fields (x400) per tumor were examined, and >300 cells were counted in each tumor. In M2TRB3 and M2mock tumors, the longest diameter of the nucleus was determined by image analysis using Olympus DP70 system (Olympus Corp., Tokyo, Japan). Four high power fields (x400) per tumor were examined and more than 100 nuclei were counted in each tumor.

These assays were performed as previously described (16). M2TRB3 and M2mock cells (7.5×10⁴ cells/plate) were plated onto 9-cm culture dishes in DMEM plus 10% FBS and grown to yield 50–60% confluence. To synchronize cells at the G0/G1 phase, they were starved by culturing in DMEM plus 0.5% FBS for 48 h. After starvation, cells were then growth in DMEM plus 10% FBS for 72 h. Adherent cells were washed twice with PBS, fixed with 5 ml 70% ethanol, centrifuged, resuspended in 400 μl PBS containing 2 mg/ml RNase (Nacalai Tesque, Inc., Kyoto, Japan), and stained with 400 μl of 0.1 mg/ml propidium iodide (Sigma-Aldrich, St. Louis, MO, USA) in the dark for 30 min or overnight. The cell suspension was filtered through a 60-μm nylon filter (Ikemoto Scientific Technology Co., Ltd., Tokyo). Samples of 10,000–20,000 cells were then analyzed for cell cycle phase distribution and ploidy status using a FACSCalibur™ instrument, and the data were analyzed with the CellQuest computer program (both from Becton-Dickinson, Franklin lakes, NJ, USA) as described in a previous study (16). Cells were harvested just after starvation (0 h) and 72 h after starvation as described in Table I and Fig. 5. Each assay was repeated more than three times to confirm the results.

These assays were conducted using previously established procedures (16). Total RNA was extracted from each cell line grown in 9-cm culture dishes using Isogen (Nippon Gene, Toyama, Japan). The reaction mixture contained 4 μg of total RNA, 1 μl of 10 mM dNTP, 1 μl of random primers (both from Life Technologies, Inc.) and 7 μl of distilled water. The reaction mixture was incubated at 65°C (5 min) for denaturation, chilled on ice for 1 min and 4 μl of 5X RT buffer (Life Technologies, Inc.), 1 μl of 0.1 M dithiothreitol (DTT), 1 μl of the RNaseOut and 1 μl of Superscript^® III Reverse Transcriptase (both from Life Technologies, Inc.) were added. After the addition of these reagents, the reaction mixture was incubated at 50°C (60 min) for random primer annealing and 70°C (15 min) for cDNA preparation. One microliter of the reaction mixture was then used for PCR. The primer sequences used in this study were as follows: human TRB3-specific primer set, hTRB3F (5′-CAAGTCGCTCTGAAGGTTCC-3′) and hTRB3R (5′-CCATCCTACTCTGGCAAAGC-3′), mouse TRB3-specific primer set, mTRB3F (5′-CAAGTCGCTCT GAAGGTTCC-3′) and mTRB3R (5′-CCATCCTACTC TGGCAAAGC-3′), respectively.

β-actin-specific DNA fragments from the same RNA samples were amplified and served as internal controls. Primers actinF (5′-CCGTAAAGACCTCTATGCCAACA-3′) and actinR (5′-CGGACTCATCGTACTCCTGCTT-3′) were used for amplification of β-actin. PCR was conducted for 26–30 cycles in an iCycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Each amplification cycle consisted of 0.5 min at 94°C for denaturation, 0.5 min at 60°C for primer annealing, and 1 min at 72°C for extension. After PCR amplification, the DNA fragments were stained with ethidium bromide and analyzed by 2% agarose gel electrophoresis. The results were confirmed by repeating the experiments.

These assays were conducted according to previously established procedures (17). The cells were lysed in radioimmunoprecipitation assay (RIPA) buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 0.5% deoxycholate, and 1% Triton X-100]. The lysates were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) (12.5%), transferred onto a polyvinylidene difluoride (PVDF) membrane (Immobilon P; Millipore Corp., Bedford, MA, USA) and probed with the antibodies. The primary antibodies used in the present study were anti-β-actin monoclonal antibody (AC-15) (Sigma-Aldrich), anti-cyclin B1 monoclonal antibody (sc-245) (Santa Cruz Biotechnology Inc.), anti-Cdc2 monoclonal antibody (sc-54), anti-Cdk2 polyclonal antibody (sc-163), anti-Cdk4 polyclonal antibody (sc-260), anti-TRB3 polyclonal antibody (sc-34211), anti-cyclin D1 monoclonal antibody (556470; Becton-Dickinson), and anti-Flag monoclonal antibody (018-22381) (Wako Pure Chemical Industries, Ltd.). The immunoreactive proteins were visualized using ImmunoStar Zeta (Wako Pure Chemical Industries, Ltd.) and light emission was quantified with Light Capture (ATTO Corp., Tokyo, Japan). Each assay was repeated more than three times to confirm the results.

Differences in the number of cells, tumor volume, PCNA labeling index, and rate of DNA ploidy between cell lines or tumor origins were analyzed by the Student’s or Welch’s t-test. A value of P<0.05 was considered to indicate a statistically significant result.

To examine the role of TRB3 in cell proliferation, we developed a cell line (M2TRB3) that stably expresses the human TRB3 gene by transfecting the gene into murine mammary tumor cell line Cl66M2 (M2) (18). We also developed the control cells (M2mock) transfected with empty vector pcDNA3.1-Hygro. M2TRB3 cells expressed both human TRB3 mRNA and mouse TRB3 mRNA (Fig. 1A). Human embryonic kidney cell line HEK293 and human hepatoma cell line HepG2 also expressed human TRB3 mRNA. There was no mRNA expression of human TRB3 in the M2 and M2mock cells. Human TRB3 DNA was PCR-amplified from pcDNA3.1-Hygro-flag-human TRB3 vector and the band was present in the positive lane in Fig. 1A. Expression of the exogenous TRB3 protein (Flag) was present in the M2TRB3 cells. No expression was noted in the protein samples derived from M2, M2mock, HEK293 and HepG2 cells (Fig. 1B). The M2TRB3 and M2mock cells were used for cell proliferation assays.

To examine the tumorigenic activity of the TRB3 gene, we investigated its effects on cell and tumor growth using M2TRB3 and M2mock cells. The number of M2TRB3 cells significantly increased compared to M2mock cells at the 48 and 72 h time points (P<0.01 and 0.05, respectively) (Fig. 2A). At 72 h, a 34% increase was noted in the number of M2TRB3 cells compared to that of the M2mock cells. To examine the extent of M2TRB3 cell growth when these cells were implanted into the subcutaneous tissue of mice, we used the xenograft mouse model as described in Materials and methods. Twice a week observation was carried out with all mice throughout the experiment. Tumor growth was monitored with the naked eye from day 7 to 35 after inoculation; tumor volume (mm³) and body weight (g) were measured twice weekly. No specific physical and behavioral changes were noted in any mice. The average volume of the tumors derived from the M2TRB3 cells was significantly increased by 38% when compared with that of the M2mock tumors at experimental day 35 (P<0.05) (Fig. 2B).

Due to the growth enhancing effects identified above, we aimed to determine whether the TRB3 gene induced morphological changes in tumor tissues. The M2TRB3, M2mock and M2 tumors were excised from the mouse skin, fixed with 10% buffered formalin, and stained with hematoxylin and eosin for histological examination. The tumors were analyzed using a light microscope. M2TRB3 and M2mock tumors presented a pedunculated round shape. Histologically, M2mock tumor cells grew in a solid appearance (Fig. 3, left upper panel). A site of necrosis was present in the central region of the tumor. M2TRB3 tumors were also solid and papillary growth was partially noted (Fig. 3, right upper panel). In the M2TRB3 tumors, the mean diameter of the nucleus (9.4±0.3 μm) was significantly greater than that (7.0±0.2 μm) of the M2mock tumors (P<0.001). The cell proliferation rate was evaluated by measuring the PCNA labeling index as described in Materials and methods. The PCNA labeling index of the M2TRB3 tumors was higher than that of the M2 and M2mock tumors but this difference was not statistically significant (Fig. 3 lower panels and Fig. 4).

Due to the differences in tumor morphology noted in Fig. 3, we examined the effects of the TRB3 gene on DNA ploidy in M2mock and M2TRB3 cells. After synchronizing cells in the G0/G1 phase, we conducted experiments at 0 and 72 h using flow cytometric analysis. Representative DNA histograms of the analysis for these cells are shown in Fig. 5A, and distribution and rate of DNA ploidy are shown in Table I. In M2mock cells, the average percentage of diploid nuclei measured 12–16%. In contrast, no diploid nuclei were observed in M2TRB3 cells (Table I and Fig. 5A, far right panels). M2TRB3 cells showed a significant increase by 19 and 12% in the population of octaploid nuclei at 0 and 72 h, respectively, when compared to M2mock cells (Table I). There was also an increase (4–19%) in the population of tetraploid nuclei in the M2TRB3 cells. These results indicate that TRB3 affects the status of DNA ploidy in mouse mammary tumor cells. M2mock cells exhibited population peaks of aneuploid nuclei (2N, 4N and 8N), indicating that these cells harbor a variable number of chromosomes.

Due to the growth enhancing effects of TRB3 as noted in Fig. 2 and nuclear hyperploidy in M2TRB3 cells, we examined whether these cells affected the levels of expression of TRB3 and cell cycle control molecules. Thus, we measured the protein expression levels of TRB3 and cell cycle control molecules cyclin B1, cyclin D1, Cdc2, Cdk2 and Cdk4. In M2TRB3 cells, both exogenous and endogenous TRB3 were highly expressed at 72 h compared to M2mock cells that only expressed endogenous TRB3 (Fig. 5B, right two columns). In contrast, a weak expression level of endogenous TRB3 was observed at 0 h in the M2mock cells, and marginal expression was noted in both exogenous and endogenous TRB3 at the same time point in these cells. Cyclin B1 and cyclin D1 expression levels in the M2TRB3 cells increased at 72 h of the cell culture, in which tumor cells were out of synchrony, compared to those of the M2mock cells (Fig. 5B). Expression levels of Cdc2, Cdk2, and Cdk4 showed no change between the M2TRB3 and M2mock cells.