Cell cycle regulation by the Wee1 Inhibitor PD0166285, Pyrido [2,3-d] pyimidine, in the B16 mouse melanoma cell line

B16 mouse melanoma cells were maintained in Dulbecco's modified Eagle's medium (DMEM); Life Technologies, (Grand Island, NY) supplemented with 10% fetal bovine serum, (FBS).

Cell proliferation was analyzed by counting the cells. B16 cells (1 × 10⁵ cells in 100 mm-dishes) were maintained in medium overnight. In addition, the cells were treated with (0, 0.5, 1.0, or 2.0 μM) PD0166285 (including DMSO vehicle) for indicated times. The cells were washed twice with phosphate-buffered saline (PBS). Next, the cells were trypsinized, so the cell numbers in each dish were determined by using a computed cell-counter (Sysmex CDA-500) according to manufacturer's recommendation.

The Wee1 inhibitor, PD0166285, was kindly provided by Pfizer (Ann Arbor, MI).

After treatment with 0.5 μM PD0166285, B16 cells were trypsinized, washed with PBS, and incubated in 0.2% Triton X-100 for 20 min at 37°C. After incubation, cells were treated with propidium iodideide (PI) and then subjected to DNA content analysis. PI florescence was analyzed with a FACScalibur flow Cytometer (Becton Dickinson). Findings characterizing flow for at least 20,000 cells were collected and analyzed with Cell Quest software (Becton Dickinson). Cell cycle distributions were calculated with Mod Fit LT software (Becton Dickinson).

Cells were incubated in glass-bottom microwell dishes (poly-d-Lysine coated, 35 mm) for at least 4 hours, with or without 0.5 μM PD166285. α-tubulin: Cells were washed with PBS, fixed in 4% paraformaldehyde in PBS for 20 min at room temperature, and permeabilized in 0.2% TritonX-100 for 10 min at room temperature. After treatment against nonspecific binding with blocking protein (DAKO, Carpenteria, CA) for 20 min at room temperature, cells were incubated with anti-α-tubulin antibody (1:400) overnight at 4°C and then with FITC-conjugated secondary antibody for 1 hour at room temperature. After washing with PBS, cells were incubated with PI for 5 min at room temperature to stain the nucleus. Observation was carried out with a laser-scanning confocal immunofluorescence microscope (Olympus, Tokyo, Japan). Wee1: Cells grown in glass bottom microwell dishes (poly-d-Lysine coated, 35 mm) were fixed in 50:50 (vol/vol) methanol/acetone and incubated with anti-Wee1 antibody at a 1:250 dilution overnight at 4°C. FITC-conjugated secondary antibody was then added for 1 hour at room temperature. Cells were counter stained with PI or double-stained with anti-α-tubulin as observed above.

Cells were lysed in lysis buffer (50 mM HEPES at pH 7.5, 250 mM NaCl, 20 mM EDTA, and 0.1% Nonidet P-40) additionally containing 1 mM phenylmethylsulfonyl fluoride, a protein inhibitor cocktail (Sigma, St. Louis, MO), 10 nM NaF, and 1 mM Na₃VO₄. Lysates were incubated at 4°C for 15 min and then cleared of cell debris by centrifugation at 14,000 g for 15 min at 4°C. Supernatants were subjected to protein determinations using a DC protein assay kit (Bio-RAD Laboratories, Hercules, CA) according to the manufacturer's instructions. Total cell lysates were then added to an equal volume of 2 × sample loading buffer containing 2% sodium dodecyl sulfate (SDS) and boiled for 5 min. Total cell protein was then loaded onto SDS-polyacrylamide gels for electrophoresis. Proteins were electrically transferred to Fluorotrans membranes for immunoblotting. The membrane was blocked for 1 hour at room temperature with 5% nonfat dry milk and then incubated with primary antibody overnight at 4°C. The membrane was washed, and incubated with horseradish peroxidase-labeled secondary antibody for 1 hour at room temperature. Finally the membrane was incubated with a detection reagent kit including luminol in an alkaline buffer, according to manufacturer's recommendations. (Amersham Pharmacia Biotech, Piscataway, NJ). Specific bands were visualized by allowing the membrane to expose blue- light-sensitive autoradiographic films.

For total RNA isolation, cultured cells were extracted using the Isogen method (Nippon Gene, Tokyo, Japan). RNA was quantified by spectrophotometry. Complementary DNA (cDNA) was synthesized using 2 μg of total RNA with random primers and Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA). The RNA and primers were denatured by heating at 70°C for 10 min. The RT reaction mixture was then incubated for 30 min at 50°C, followed by 15 min at 70°C. A template-free control was processed for each experiment, establishing an absence of genomic contamination in the samples. The resulting cDNA then was amplified by PCR (20 cycles) with primer pairs specific for cyclin D, or glyceraldehyde-3-phosphate dehydrogenase. PCR products were resolved in 1.5% agarose gels and visualized by ethidium bromide staining with ultraviolet transillumination. PCR cycle conditions were 94°C for 30 s, 6°C for 30 s and 72°C for 1 min. The primer pair sequences were as follows: cyclin D, 5'-TCCGGAGACCGGCAGTACAG-3' and 5'-TTGCAGCAGCTCCTCGGGC-3' (512 bp), and GAPDH, 5'-ACCACAGTCCATGCCATCAC-3' and 5'-TCCACACCCTGTTGCTGTA-3' (452 bp).

For quantitative SYBR Green real-time PCR, 0.6 μl of the RT products were used for each reaction, carried out using a SYBR Green PCR Core Reagent kit (PE Biosystems, Warrington, UK) according to the protocol provided by the manufacturer. Optimal conditions were established for each specific gene. Primer sequences were designed for cyclin D as follows using Primer Express Software (PE Applied Biosystems, Foster City, CA): 5'-GCAGCACCCGGTCGTTGAGGA-3' and 5'-TCCGGAGACCGGCAGTACA-3' (180 bp). After quantitative PCR was performed, products were analyzed using the ABI PRISM 7000 Sequence Detection System (PE Applied Biosystems). A three-stage PCR cycle program provided by the manufacturer was used, as follows: 2 min at 50°C, 10 min at 95°C, and then 40 cycles of 15 s at 95°C and 1 min at 60°C. Reaction products were visualized on ethidium bromide-stained 1.5% agarose gels. Appearance of a single band of the correct molecular size confirmed PCR specificity. Results are representative of three independent experiments [20].

Cell proliferation was analyzed by counting the cell numbers. We counted the cell number with DMSO control, 0.5, 1, and 2 μM treatment for 4 hours and 24 hours. 0.5 μM of PD0166285 was bottom line to anti-proliferate (Figure 1A). Cell cycle changes were investigated using flow cytometry. B16 cells were arrested in the early G1 stage after treatment with 0.5 μM PD0166285. No cells showing characteristics of apoptosis were observed (Figure 1B). The percentage of cells in the G0/G1 phase was 67% at 0 hours after PD0166285 treatment, and 86% at 4 hours. The percentage in the S phase was 16% at 0 hours, and 12% at 4 hours. The percentage in the G2 phase was 17% at 0 hours, and 2% at 4 hours. S-, G2-, and M-phase cells progressed to the early G1 phase within 4 hours. PD0166285 thus promoted complex cell cycle progression from G2 to the G1 phase in B16 cells. At 24 hours, the G1, S and G2 cell distributions were 81, 15 and 4%, respectively after PD0166285 treatment with 0.5 μM. In DMSO control, the percentage of cells was almost same within 0 h to 24 h. Sub-G1 which means that apoptosis was absent at 24 hours.

Double-staining immunofluorescence microscopy with PI (red) and anti-α-tubulin (green) after 4 hours of PD0166285 treatment is shown in Figure 2A. Nuclei in most of the cells had become small and the cell number had increased. However, the cells did not spread. Typical interphase microtubule networks had disappeared at 4 hours. Photomicrographs at 0 hours, 2 hours, and 4 hours show disappearances of microtubule networks in those cells not exhibiting mitosis (Figure 2B). PD0166285 thus inhibited microtubule stabilization. The amount of α-tubulin protein in cells with and without PD0166285 treatment remained the same (DATA not shown).

PD0166285 almost completely abolished cdc2-Tyr15 phosporylation in B16 cells (Figure 3A). Expression of cdc2 were no change, but the expression of cyclin B decreased. We then characterized the cellular localization of cyclin B by laser confocal immunofluorescence microscopy. At 4 hours, cyclin B expression was diminished, although it could be seen in most cells in the early G1 phase (Figure 3B)

We characterized Wee1 in terms of its subcellular location in the cytoplasm, and also changes evident upon cell adhesion. Without PD0166285 treatment, Wee1 was seen extensively in the cytoplasm. Upon PD0166285 treatment, Wee1 was clustered tightly around nuclei while cells appeared shrunken (Figure 4A). Wee1 and α-tubulin were cellular co-localization molecules. After treatment, they were restricted in distribution. The spreading of cell and plasma membrane ruffling were suppressed in morphogenesis (Figure 4B). This indicated imperfect adhesion.

To address the mechanisms of cell cycle arrest induced by PD0166285 in B16 cells, expression of endogenous CDK inhibitors (CKIs) such as p16, p21, and p27 were investigated by immunoblot analysis with specific antibodies for CKIs (Figure 5A). P16 protein was not detected. P21 and p27 were detectable, but amounts were unchanged at also 24 houres (Figure 5B). Progression through the cell cycle is governed by kinase activities of specific CDKs, which are regulated by association with the regulatory cyclin subunits. We investigated cyclin D and E, which are related to the progression from the G1 to the S phase. The progression through the G1 phase involves mainly CDK4/6-cyclin D in the early G1 phase and CDK2-cyclinE in the mid G1 phase. Cyclin D decreased gradually until 4 hours. Cyclin E was found to decrease at 2 hours, but then it somewhat increased at 4 hours. P16, an inactivator of CDK4/6-cyclin D, was not detected in B16 cells. Therefore, it is known that cell cycle arrest is the suppression of cyclin D expression in B16 cells by PD0166285. We next investigated Rb, which regulates cell cycle progression from G1 to the S phase via E2F after treatment for 0, 4 and 24 hours. An immunoblot analysis showed a gradually inactive dephosphorylation of Rb, (Figure 5B).

We next investigated cyclin D mRNA transcription, using RT-PCR. Cyclin D mRNA transcription decreased after 4 hours of treatment with PD0166285 (Figure 6A). We then examined the transcription of cyclin D mRNA more quantitatively by real-time PCR. Expression of cyclin D mRNA decreased to 42.1% of baseline levels at 4 hours (Figure 6B). Thus, PD0166285 down-regulated the transcription of cyclin D mRNA within 4 hours, a time course similar to that of microtubule destabilization.