The cloning of cyclin D1b complementary DNA and construction of the expression plasmids, pCR3.1-cyclin D1b-Flag and pCR3.1-cyclin D1a-Flag, and pCR3.1-cyclin D1-T286A-Flag were performed as previously described (13).

A tumor cell line, D1bTgRT, was established from a rectal tumor of a cyclin D1b Tg mouse. D1bTgRT cells were cultured in RPMI-1640 medium (Nacalai Tesque, Inc., Kyoto, Japan) supplemented with 10% fetal calf serum (FCS) (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany), penicillin (100 U/ml) and streptomycin (100 µg/ml) in humidified air containing 5% CO₂ at 37°C. MEF cells prepared from the embryos of WT and cyclin D1b Tg mice were immortalized by the large T antigen of Simian vacuolating virus 40 (SV40) (14). Furthermore, MEF cells were infected with retrovirus expressing activated K-ras, known as pBABEpuro-K-Ras (21). 293T cells derived from human embryonic kidney were used for plasmid transfection. These cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Nacalai Tesque, Inc.) supplemented with 10% FCS, penicillin (100 U/ml) and streptomycin (100 µg/ml) in humidified air containing 5% CO₂ at 37°C.

Cell proliferation assay was performed by culturing the cells in 35-mm culture dishes. A total of 2×10⁴ of the cells were cultured in triplicate into each dish and incubated at 37°C. Viable cells were trypsinized and counted using a hemocytometer.

The in vitro cell invasiveness was evaluated by a Matrigel™ Basement Membrane Matrix Invasion Chamber (chamber size, 6.4 mm; membrane surface area, 0.3 cm²; pore size, 8 µm; BD Biosciences, Franklin Lakes, NJ, USA), according to the manufacturer's protocol (13). A total of 500 µl of cell suspension (5×10⁴ cells/ml) was added to each chamber. The chambers containing the cells were incubated for 4 days in a CO₂ (5%) incubator. Noninvasive cells were removed from the upper surface of the membrane. The invasive cells on the underside were stained with Diff-Quik™ stain (Kokusai-Shiyaku, Kobe, Japan) and counted under a microscope (TMS; Nikon Corporation, Tokyo, Japan). Each cell sample was evaluated in triplicate.

Anchorage-independent growth of the cells was evaluated by colony-forming ability in soft agar (13). A total of 10,000 cells were inoculated into a 60-mm dish containing 0.4% Noble agar containing DMEM supplemented with 10% FCS and incubated at 37°C. The number of colonies (>0.15 mm in diameter) on each plate was scored after 3 weeks of incubation. Each assay was performed in triplicate.

Tumorigenicity of the cells was examined by subcutaneous injection of 3×10⁶ cells per 0.2 ml PBS (−) into 6-week-old BALB/c-nu/nu female nude mice (20 g). The animals were housed in a specific pathogen-free room with controlled temperature (20–22°C), humidity (50–60%) and a pre-set light-dark cycle (12:12 h). They were allowed ad libitum access to food (CE-2; CLEA Japan, Inc., Tokyo, Japan) and water. A total of four weeks subsequent to injection, the tumorigenic potential of the cells was assessed by measuring the weight and volume of tumors. The use of the animals in the experimental protocols was reviewed and approved by the Committee of Research Center for Animal Life Science at Shiga University of Medical Science (Otsu, Japan; approval no. 2014-12-5).

Transfection of plasmid DNA was performed using Lipofectamine™ PLUS reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer's protocol. Briefly, 5×10⁵ cells were inoculated into a 60-mm dish and, upon overnight incubation at 37°C, 2 µg of plasmid DNA was transfected.

Lysates of cells and tissues were prepared by Laemmli-sodium dodecyl sulfate (SDS) buffer containing 62.5 mM Tris-HCl (pH 6.8), 10% glycerol, 5% 2-mercaptoethanol, 2% SDS, 0.01% bromophenol blue and 5 mM ethylenediaminetetraacetic acid. Each cell lysate (20 µg of protein) underwent 10 or 12% SDS-PAGE, and the proteins were electrotransferred to Immobilon-P membranes (EMD Millipore). Following blocking with TBS-T [10 mM Tris-HCl (pH 7.6), 150 mM sodium chloride and 0.1% Tween-20] containing 5% bovine serum albumin (BSA) (Nacalai Tesque, Inc.), the membranes were incubated with the primary antibodies diluted 1:1,000 in TBS-T containing 2% BSA. Following washes with TBS-T, the membranes were incubated for 1 h at room temperature in horseradish peroxidase-conjugated anti-mouse or or anti-rabbit immunoglobulin G (NA931 and NA934, respectively; GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) diluted 1:20,000 in TBS-T containing 2% BSA. Following washing with TBS-T, immunoreactivity was detected by an enhanced chemiluminescence system (GE Healthcare Bio-Sciences) using X-ray films.

The primary antibodies used for immunoblotting were as follows: Anti-α-tubulin (DM1A) (T9026) and anti-FLAG (M2) (F3165) monoclonal antibodies were obtained from Sigma-Aldrich (Merck Millipore). Anti-phospho Akt (T308 and S473, and D25E6 and D9E) (#13038 and #4060, respectively) rabbit monoclonal, anti-Akt rabbit polyclonal (#9272), anti-phospho Erk1/2 (p44/42 MAPK) rabbit monoclonal (T202/Y204, D13.14.4E) (#9101), anti-Erk1/2 (p44/42 MAPK) rabbit monoclonal (137F5) (#4695), anti-phospho-MEK1/2 rabbit monoclonal (S221, 166F8) (#16211), anti-MEK1/2 rabbit monoclonal (47F6) (#9126) and anti-cyclin D1 rabbit monoclonal (92G2) (#2978) primary antibodies, which were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Anti-E-cadherin (#610181) and anti-N-cadherin (#13116) monoclonal primary antibodies were purchased from BD Biosciences, and the anti-vimentin (C9) (sc-6260) mouse monoclonal antibody was obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). All antibodies were used at a dilution of 1:1,000.

The use of the animals in the experimental protocols was reviewed and approved by the Ethics Committee of the Research Center for Animal Life Science at Shiga University of Medical Science (Otsu, Japan). The right dorsal flank of each 6-week-old BALB/c-nu/nu female nude mouse was injected subcutaneously with 3×10⁶ D1bTgRT cells. Following, establishment of palpable tumors (>100 mm³), external tumor volume was determined on days 0, 7 and 11. A total of six mice carrying palpable tumors were randomized into two groups of three mice each. Each Akt inhibitor at a dose of 25 pmol/mg (calculated tumor weight) or a similar volume of dimethyl sulfoxide (DMSO) was injected into the tumor of each mouse on days 0 and 7. Tumor volume was calculated using the formula: AxBxC/2, where A, B and C represent the diameters of length, height and width, respectively. The volume of injection for a tumor was prepared as 100 µl.

Using the paired Student's t-test, the present study compared the invasiveness of D1bTgRT cells treated with Akt inhibitor with those treated with DMSO as a control, and the in vivo tumorigenicity of D1bTgRT cells treated with Akt inhibitor with those treated with DMSO as a control, in the same nude mouse. P≤0.05 was considered to indicate a statistically significant difference. All statistical analyses were performed using the R statistical software package, version 2.6.2 (The R Project for Statistical Computing, Vienna, Austria).

To investigate the role of cyclin D1b in cell transformation and the signaling pathway resulting in Erk activation, the present study established WT and cyclin D1b-expressing MEF cell lines, named WT-LT and D1b-LT, respectively, by introducing the large T antigen of SV40 into embryonic fibroblasts derived from WT and cyclin D1b-Tg mouse. Significant differences were not observed between WT-LT and D1b-LT cells in terms of cell proliferation (Fig. 1A and B). However, D1b-LT cells had a slightly more spindle-shaped appearance compared with WT-LT cells. Subsequently, to investigate whether the expression of cyclin D1b elevates the sensitivity to in vitro cell transformation, an activated K-ras oncogene was introduced into the cell lines using the retrovirus vector pBABE-puro and established vector and K-ras-expressing WT-LT and D1b-LT cell lines, named WT-LT/BP, WT-LT/K-ras, D1b-LT/BP and D1b-LT/K-ras. The morphology of D1b-LT/K-ras cells was significantly altered, although the morphology of the WT-LT/K-ras cells was also transformed into a more spindle-like appearance, as compared with D1b-LT cells (Fig. 1A). The proliferation of D1b-LT/K-ras cells was increased compared with any of the other cells (Fig. 1B). The present study subsequently investigated the anchorage-independent growth of these cells; as shown in Fig. 1C, D1b-LT/K-ras cells produced a large number of colonies in soft agar, whereas WT-LT/BP cells scarcely produced any colonies and WT-LT/K-ras and D1b-LT/BP cells slightly produced colonies. The present study also investigated the tumorigenicity of these cells in nude mice. As shown in Fig. 1D, although WT-LT/K-ras and D1b-LT/K-ras cells demonstrated tumorigenicity in nude mice, the tumor volumes of the D1b-LT/K-ras cells were significantly increased compared with that of the WT-LT/K-ras cells (P=0.039). Neither WT-LT/BP nor D1b-LT/BP cells produced tumors in nude mice. WT-LT/K-ras and D1b-LT/BP cells showed a more invasive phenotype compared with WT-LT cells in the Matrigel assay, and expression of cyclin D1b and K-ras synergistically enhanced cell invasiveness (Fig. 1E). These results indicate that cyclin D1b by itself has weak transformation activity, and cyclin D1b-expressing MEF cells are more sensitive to transformation by the activated K-ras oncogene than WT MEF cells, suggesting that cyclin D1b cooperates with activated K-ras to transform MEF cells.

As cyclin D1b enhanced cell invasiveness, the present study investigated alterations to the expression of proteins involved in the epithelial-to-mesenchymal transition and Akt phosphorylation by immunoblot analyses with specific antibodies. As shown in Fig. 2A, expression of E-cadherin was downregulated in WT-LT/K-ras and D1b-LT/BP cells compared with that in WT-LT/BP cells; its expression was completely lost in D1b-LT/K-ras cells. This result is compatible with the morphological alterations observed in transformed MEF cells (Fig. 1A). The levels of N-cadherin and vimentin were similar in all cell lines. Phosphorylation of Akt (T308 and S473 residues) was enhanced in WT-LT/K-ras, D1b-LT/BP and D1b/K-ras cells when compared with WT-LT/BP cells. The levels of total Akt and α-tubulin protein were similar in all cell lines. These results indicate that cyclin D1b is able to suppress E-cadherin expression and to activate Akt in MEF cells. To clarify whether Akt activation is necessary for an increase of cell invasiveness by cyclin D1b, a Matrigel invasion assay of WT-LT and D1b-LT MEF cells treated with an Akt inhibitor (25 µM) was performed. As shown in Fig. 2B, the Akt inhibitor significantly suppressed the invasiveness of D1b-LT MEF cells (P=0.0116), but not of WT-LT MEF cells (P=0.9947), indicating that Akt activation has a significant role for the enhancement of cell invasiveness in cyclin D1b-expressing MEF cells.

To clarify the role of Akt in the rectal tumorigenicity of cyclin D1b Tg mice, the present study examined the phosphorylation of Akt in normal rectum and rectal tumors (SSA/Ps and adenocarcinomas) of cyclin D1b Tg mice. As shown in Fig. 3A, phosphorylation of Akt was increased in rectal tumors as compared with normal rectal tissue. As previously reported, Erk was also activated in rectal tumors without activation of MEK (14). Although the present study also examined the expression of E-cadherin in normal rectum and rectal tumors, alteration of its expression was not observed in the rectal tumors of these Tg mice (Fig. 3B). The present study also investigated Akt activation in 293T cells transfected with cyclin D1b, cyclin D1a and cyclin D1-T286A. Cyclin D1-T286A is a mutant of cyclin D1a, in which threonine-286 (T286), the site of glycogen synthase kinase-3β phosphorylation that is required to enhance both the nuclear export of cyclin D1 and its turnover, is replaced with alanine (22). The mutation of T286 to a nonphosphorylatable residue promotes a constitutively nuclear localization of the cyclin D1a protein, with enhanced oncogenic potential (23). As shown in Fig. 3C, increased expression of cyclin D1b and cyclin D1-T286A by transfection accelerated the phosphorylation of Akt, suggesting that nuclear localization of these cyclin D1 proteins is critical for Akt activation. The present authors have previously reported that expression of cyclin D1b increases Erk phosphorylation without activating MEK in 293T cells (14). To clarify whether activation of Erk by cyclin D1b is mediated through Akt activation, the present study investigated the effects of an Akt inhibitor on Erk activation by cyclin D1b in 293T cells. As shown in Fig. 3D, treatment with an Akt inhibitor suppressed the enhanced phosphorylation of Erk and Akt in 293T cells expressing cyclin D1b. The present study also investigated the effects of the Akt inhibitor on Erk activity in D1bTgRT cells from a rectal tumor of the cyclin D1b Tg mouse. As shown in Fig. 3E, treatment of the Akt inhibitor suppressed phosphorylation of Erk and Akt in D1bTgRT cells. These results show that cyclin D1b activates Erk via Akt to contribute to tumor formation.

To clarify the role of Akt in the cell invasiveness of rectal tumor cells expressing cyclin D1b, the present study examined the effect of an Akt inhibitor on the cell invasiveness of D1bTgRT cells. As shown in Fig. 4A, treatment with the Akt inhibitor (25 µM) significantly reduced the cell invasiveness of D1bTgRT cells (P=0.0007). Furthermore, the present study examined the effect of Akt inhibition on tumor growth in nude mice. As shown in Fig. 4B, the tumor growth of D1bTgRT cells was significantly decreased by Akt inhibitor treatments (P=0.0425). These results indicate that enhanced phosphorylation of Akt by cyclin D1b contributes to cell invasiveness and tumor formation of the rectal tumor cell line, D1bTgRT.