Four human colorectal carcinoma and matched adjacent normal colorectal tissue samples were collected in the Third Hospital of Jilin University (Changchun, China) between January, 2010 and December, 2010, in accordance with the protocols approved by the Institutional Review Board of the Third Hospital of Jilin University. Patients provided written informed consent for the tissue collection. This study was approved by the Third Hospital Ethics Committee of Jilin University.

The colorectal carcinoma cell lines CACO-2, COLO-205, COLO-320, DLD-1, HCT-8, HT29 and SW948, together with the human glioma cell line LN229 serving as the control, were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Each cell line was grown in RPMI-1640 medium (Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) and maintained in a humidified 37°C and 5% CO₂ incubator (26).

PD-0332991 was purchased from Selleckchem (Houston, TX, USA) and prepared as stock solutions in dimethyl sulfoxide (DMSO). Antibodies against CDK1, CDK4, CDK6, cyclin D1, cyclin D3, RB, phosphorylated-RB (pRB) (S780, S795 and S807/811) were purchased from Cell Signaling Technology, Inc., (Danvers, MA, USA). The antibody against CDK2 was purchased from Santa Cruz Biotechnology, Inc., (Santa Cruz, CA, USA) and β-actin antibody was purchased from Sigma-Aldrich (St. Louis, MO, USA). Horseradish peroxidase-conjugated goat anti-mouse and goat anti-rabbit antibodies were obtained from Jackson IR Laboratories, Inc., (West Grove, PA, USA). Protease inhibitor mixture, Triton X-100 and other chemicals were purchased from Sigma-Aldrich. The enhanced chemiluminescence detection kit was obtained from Amersham Biosciences (Piscataway, NJ, USA).

Western blotting was performed as previously described (27). In brief, cell lines and tissues were lysed in lysis buffer consisting of 20 mmol/l Tris pH 7.4, 150 mmol/l NaCl, 1% NP-40, 10% glycerol, 1 mmol/l EGTA, 1 mmol/l EDTA, 5 mmol/l sodium pyrophosphate, 50 mmol/l sodium fluoride, 10 mmol/l β-glycerophosphate, 1 mmol/l sodium vanadate, 0.5 mmol/l DTT, 1 mmol/l PMSF, 2 mmol/l imidazole, 1.15 mmol/l sodium molybdate, 4 mmol/l sodium tartrate dihydrate and 1X protease inhibitor cocktail (Sigma, St. Louis, MO, USA). Following a 30-min incubation in lysis buffer at 4°C, lysates were centrifuged at 18,000 × g for 15 min at 4°C. The supernatant was collected and protein concentrations were determined by the Bradford protein assay following the manufacturer’s instructions (Bio-Rad, Hercules, CA, USA). Equal amounts of protein were separated through SDS-PAGE gels and transferred onto nitrocellulose membranes (Bio-Rad). The membranes were incubated overnight at 4°C with primary antibody and then for 1 h with horseradish peroxidase-conjugated secondary antibody. The membranes were developed by chemiluminescence.

Lentiviral shRNA vectors were purchased from the Sigma MISSION^® shRNA library (Sigma-Aldrich) and included scrambled control (SHC002), CDK6-747 (TRCN0000039747, 5′-CGTGG AAGTTCAGATGTTGAT-3′) and CDK6-893 (TRCN000019 4893, 5′-CATGAGATGTTCCTATCTTAA-3′), CDK4-20 (TRCN0000010520, 5′-ACAGTTCGTGAGGTGGCTTTA-3′), and CDK4-64 (5′-ATGACTGGCCTCGAGATGTAC-3′). Each lentiviral shRNA vector was transduced into cells that were selected with puromycin as previously described (28).

Cell proliferation was determined by acid phosphatase assay according to the manufacturer’s instructions (29,30). In brief, untreated or transduced cells with shRNA vectors were grown in 96-well plates at 1×10³ cells per well in 200 μl of 10% FBS-containing medium. Following incubation for 24 h, the medium was replaced with 10% FBS medium or the medium was supplemented with PD-0332991. After incubation for 1, 3 or 5 days, the cells were washed with phosphate-buffered saline (PBS) and each of the wells was added with 100 μl buffer containing 0.2 M sodium acetate (pH 5.5), 0.2% (v/v) Triton X-100 and 20 mmol/l p-nitrophenyl phosphate (Sigma 104 phosphatase substrate). The plates were incubated at 37°C for 1.5 h and the reaction was stopped by the addition of 10 μl 1 M NaOH to each well and staining was measured at 405 nm by a microplate reader (Bio-Rad).

Flow cytometry was performed as previously described (26). In brief, cells were grown in 65-mm plates at a density of 5×10⁵ cells per well. After 24 h incubation, the cells were grown for 24 h in 10% FBS medium supplemented with or without PD-0332991 (1 μmol/l) in the presence or absence of DMSO as a control. After treatment, cells were collected, washed with PBS and fixed by incubation in 70% ethanol solution at 4°C. The fixed cells were washed and the cell pellets were stained using a propidium iodide-RNase solution (PBS containing 20 μg/ml propium iodide, 20 μg/ml DNase-free RNase A and 0.1% Triton X-100) for 30 min at 20°C in the dark. The cell cycle status was analyzed with a flow cytometer using FlowJo software (Tree Star, Inc., Ashland, OR, USA).

Data were presented as the means ± standard deviation (SD) and analyzed statistically by Student’s t-test. P<0.05 was considered statistically significant.

To the best of our knowledge, this is the first study concerning the expression of cell cycle proteins in colorectal carcinoma tissues. We examined the expression of key cell cycle proteins in surgically resected colorectal carcinoma tissues as compared with matched adjacent normal colorectal tissues. Western blotting revealed that CDK1, CDK2, CDK4 and CDK6 proteins were expressed at much higher levels in the carcinoma tissues than the matched normal tissues (Fig. 1A). These CDK proteins were expressed in the carcinoma-derived cell lines (Fig. 1B). Cyclin D1 was detected in half of the carcinoma tissues and cyclin D3 was observed in only one of the four carcinoma tissues, while cyclin D1 and D3 were slightly detected in normal tissues. By contrast, cyclin D2 was expressed in all the carcinoma and matched normal tissues with the expression levels being higher in the carcinoma tissues. Consistent with this profile, cyclin D1 was highly expressed in four but weakly in three; cyclin D2 was highly expressed in three and cyclin D3 was expressed in seven of eight cell lines.

We examined the expression of unphosphorylated RB and pRB. RB can be phosphorylated at several serine residues including S780, S795, S807 and S811 (31). Thus, the antibodies against pRB (S780), pRB (S795) and pRB (S808/811) were used in this study. Western blotting detected pRB (S780) and pRB (S808/811) in three of four carcinoma tissues but only slightly in the normal tissues (Fig. 1A). The pRB (S795) was observed only in the cell lines but not any tissues as compared with the expression in the control glioblastoma cell line LN229. By contrast, pRB (S795) was detected in all the cell lines (Fig. 1B). Collectively, the G1 cyclins, CDK and pRB are expressed differentially in human colorectal carcinoma tissues and cell lines.

To explore the potential of PD-0332991 in treating colorectal carcinoma, we examined whether the carcinoma cells respond to PD-0332991 treatment in culture. The carcinoma cell lines DLD-1 and COLO320 were treated with PD-0332991 concentrations ranging between 25 nM and 5000 nM for 72 h. Results of the cell proliferation assay showed that PD-0332991 treatment significantly inhibited the growth of DLD-1 (Fig. 2A) and COLO320 cells in a dose-dependent manner (Fig. 2B). To evaluate whether inhibition by PD-0332991 occurs through the cell cycle, DLD-1 and COLO320 cells were treated or untreated with PD-0332991 (1 μM) for 48 h and subjected to flow cytometry for the cell cycle as previously described (30). The results showed that the PD-0332991 treatment led to a significant increase in the number of G1 phase cells but a marked decrease of the S phase cells in the DLD-1 (Fig. 2C) and COLO320 cells (Fig. 2D). These findings suggest that PD-0332991 treatment inhibits growth through the induction of G1 arrest of colorectal carcinoma cells.

To examine the mechanisms in the PD-0332991-induced G1 arrest, we examined the CDK4/6 and RB proteins in DLD-1 and COLO320 cells after 24 h treatment with a series of concentrations of PD-0332991. Western blotting revealed that the treatment did not affect the levels of CDK4, CDK6 and unphosphorylated RB in DLD-1 (Fig. 3A) and COLO320 cells (Fig. 3B). By contrast, PD-0332991 treatment markedly reduced the levels of pRB (S780) and pRB (S795) in each of the cell lines in a dose-dependent manner. Collectively, PD-0332991 treatment inhibits RB phosphorylation, induces G1 arrest and thus suppresses the growth of colorectal carcinoma cells in culture.

CDK4 and CDK6 regulate the G1-S cell cycle transition (2). However, whether CDK4 or CDK6 or both were required for RB phosphorylation and G1-S transition in colorectal carcinoma cells remained to be clarified. To address this issue, lentiviral vectors encoding CDK4/CDK6-specific shRNA sequences were transduced into COLO320 cells as previously described (28). To prevent off-target effects, two shRNA target sequences were used for each of the kinases including CDK4-20 and CDK4-64 to target CDK4 and CDK6-747 and CDK6-893 for CDK6 knockdown. The transduced cells were examined by western blotting and the results revealed that the transduction of the CDK4 and CDK6 shRNA encoding vectors eliminated the expression of CDK4 (Fig. 4A) and CDK6 protein (Fig. 4B), respectively, in COLO320 cells.

Notably, CDK4 knockdown did not affect RB phosphorylation as evidenced by western blotting using the pRB (S780) and pRB (S795) antibodies (Fig. 4A). By contrast, CDK6 knockdown markedly reduced the expression of these phosphorylated RB proteins in the carcinoma cells (Fig. 4B). To examine the effects of CDK4/6 knockdown and RB phosphorylation inhibition on cancer cell growth, we transduced the shRNA-coded vectors in COLO320 cells, selected stably transduced cells and examined cell growth using a cell viability assay. The results showed that the knockdown of CDK4 slightly inhibited the growth of COLO320 cells (Fig. 4C), whereas the knockdown of CDK6 resulted in a marked inhibition of the growth of COLO320 cells (Fig. 4D). The data suggest that CDK6 plays a critical role in RB phosphorylation and cell growth in colorectal carcinoma cells.