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

1471-2407-6-292 1471-2407 Research article Cell cycle regulation by the Wee1 Inhibitor PD0166285, Pyrido [2,3-d] pyimidine, in the B16 mouse melanoma cell line Hashimoto Osamu osamuhas@med.kurume-u.ac.jp Shinkawa Masako mashin@med.kureme-u.ac.jp Torimura Takuji tori@med.kureme-u.ac.jp Nakamura Toru ntoru@med.kureme-u.ac.jp Selvendiran Karuppaiyah nina@med.kureme-u.ac.jp Sakamoto Masaharu miyabi@med.kureme-u.ac.jp Koga Hironori hirokoga@med.kureme-u.ac.jp Ueno Takato takato@med.kureme-u.ac.jp Sata Michio msata@med.kureme-u.ac.jp

Liver Cancer Division, Research Center for innovatve cancer therapy and Center of the 21st century COE program for medical Science, Kurume University, Kurume, Japan

The division of Gastroenterology, Internal Medicine, Kurume University of medicine, Kurume, Japan

BMC Cancer 1471-2407 2006 6 1 292 http://www.biomedcentral.com/1471-2407/6/292 17177986 10.1186/1471-2407-6-292 13 7 2006 19 12 2006 19 12 2006 2006 Hashimoto et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Wee1 kinase plays a critical role in maintaining G2 arrest through its inhibitory phosphorylation of cdc2. In previous reports, a pyridopyrimidine molecule PD0166285 was identified to inhibit Wee1 activity at nanomolar concentrations. This G2 checkpoint abrogation by PD0166285 was demonstrated to kill cancer cells, there at a toxic highest dose of 0.5 μM in some cell lines for exposure periods of no longer than 6 hours. The deregulated cell cycle progression may have ultimately damaged the cancer cells. We herein report one of the mechanism by which PD0166285 leads to cell death in the B16 mouse melanoma cell line.

Methods

Tumor cell proliferation was determined by counting cell numbers. Cell cycle distribution was determined by flow cytometry. Morphogenesis analysis such as microtubule stabilization, Wee1 distribution, and cyclin B location were observed by immunofluorescence confocal microscopy. An immunoblot analysis of cdc2-Tyr15, cyclin D, E, p16, 21, 27, and Rb. A real-time PCR of the mRNA of cyclin D were completed.

Results

In our experiment, B16 cells also dramatically abrogated the G2 checkpoint and were found to arrest in the early G1 phase by treatment with 0.5 μM for 4 hours observed by flow cytometry. Cyclin D mRNA decreased within 4 hours observed by Real-time PCR. Rb was dephosphrylated for 24 hours. However, B16 cells did not undergo cell death after 0.5 μM treatment for 24 hours. Immnofluoscence microscopy showed that the cells become round and small in the morphogenesis. More interesting phenomena were that microtubule stabilization was blocked, and Wee1 distribution was restricted after treatment for 4 hours.

Conclusion

We analyzed the effect of Wee1 inhibitor PD0166285 described first by Wang in the G2 transition in the B16 melanoma cell line. The inhibitor PD0166285 abrogated G2/M checkpoint inducing early cell division. Moreover, we found that the treatment of cells with the inhibitor is related to microtubule stabilization and decrease in cyclin D transcription. These effects together suggest that Wee1 inhibitor may thus be a potentially useful anti-cancer therapy.

Background

The progression of the mammalian cell cycle is controlled by the sequential activation of a series of cell cycle-dependent kinases (CDKs) 1. Dysfunction of these molecular checkpoints results in the proliferation of cancer cells. In this context, an abrupt shift of the cell to mitosis from the G2 phase has received increasing attention, as have elements of the G2 checkpoint, particularly Wee1 2.

The activation of the mitosis-promoting kinase cdc2 is required for transition from the G2 to the G1 phase in all eukaryotic cells. Cdc2 is subject to multiple levels of regulation, including association with its major partner B-type cyclin, reversible phosphorylation, and intracellular compartmentalization. After association of cdc2 with cyclin B, activity of cdc2-cyclin B is repressed to a basal level until G2/M transition, when the G2/M checkpoints are complete 34. Phosphorylation of cdc2 at Thr-14 and Tyr-15 is critical in the repression of cdc2-cyclin B. The protein kinase Wee1 56 phosphorylates at Tyr-15, while another protein kinase membrane-associated cdc2 tyrosine- and threonine-specific cdc2 inhibitor (Myt1) phosphorylates both site 67. Cdc25C, on the other hand, is a phosphatase that dephosphrylates cdc2 at Thr-14 and Tyr-15. As a result cyclin B-cdc2 is activated and the cell cycle progresses. Because the Thr-14 and Tyr-15 phosphorylations are crucial for function of the G2/M checkpoint 8, induction of G2 arrest may require activation of Wee1 and Myt1 in addition to inactivation of Cdc25C 9.

Human Wee1 is inactivated through phosphorylation and protein degradation during the M phase. This degradation of Wee1, carried out through ubiquitination by cdc34 10 and the ubiquitin ligase complex (Skp1, CDC53/Cullin, F-box protein) 11, also is regulated by cdc2-cyclin B 12. Typically, irradiation-induced DNA damage favors inactivation of Cdc25C as follows. The mechanism by which Cdc25C is inactivated involves phosphorylation at Ser-215 catalyzed by Chk1/Chk2, and a 14-3-3 exportion from nuclei. The upstream kinase that activates Chk1 is ATM, which can be activated by DNA damage. Such Cdc25C inactivation helps to maintain cell cycle arrest by Wee1. Another possibly relevant pathway involves the DNA damage response kinases, checkpoint kinase (Chk1) and serine/threonine-protein kinase (Cds1), which directly phosphorylate Wee1. However, the physiologic significance of this phosphorylation remains obscure 1314.

After mitosis, daughter cells adhere to the extracelluler matrix. Cyclin D, which acts to initiate the cell cycle, then is expressed. Cyclin D expression is important for progression through the G1 phase. Expressions of cyclin D increased due to various stimuli. Initially, cyclin D is increased by the Rac-integrin signal associated with cell-to-cell matrix adhesion. After some hours, cyclin D expression is regulated through Erk signaling by growth factors 15. Cyclin D combines with CDK4/6, cyclin E/CDK2 and cyclin A/CDK2 16. The cell will then advance into the G1 phase. E2F, which regulates the trasnscription of various molecules, is activated through phosphorylation inactivating Rb by CDKs; cells then advance into the S phase 17.

PD0166285, a ppyrido [2,3-d] pyimidine compound, was developed 18 as an inhibitor of Wee1; inhibition is evident even at nanomolar concentrations. Irradiation induces DNA injury, so cell arrest which prevent cell apoptosis. This is one of the reasons that effect of inducing apoptosis to cancer cells is restricted in radiation therapy. In seven cancer cell lines, 0.5 μM of PD0166285 for short exposure period (2–4 hour) was found to dramatically inhibit the radiation-induced cdc2 phosphorylation, which otherwise would have resulted from Cdc25C inactivation. Before the G2/M transition leading to cell division, Wee1 reversibly arrests the cell cycle by inactivating cdc2 through phosphorylation at Tyr-15. PD0166285 may thus nullify the G2 checkpoint. Because Wee1 is inhibited, cancer cells do not undergo cell cycle arrest, so mitosis continues despite radiation-induced injury to DNA. However, in cells susceptible to p53 and the G1 check point killing, PD0166285 acts as a radiosensitizer promoting cell death. The sensitivity enhancement ratio was found to be 1.23 by a clonogenic assay. Interestingly, this radiosensitizing activity is p53 dependent, thus showing greater efficacy in cells where p53 is active. This inhibitor represents a novel class of anticancer drugs that selectively enhance cancer cell killing by conventional therapies.

However, PD0166285 kills tumor cells directly in some cell lines at a toxic highest dose of 0.5 μM for long exposure periods of no more than 6 hours with no relation of p53. These effects were observed in many cell lines such as ovarian carcinoma PA-1 cells, colon cancer HT29, HCT116, cervical cancer HeLa, lung carcinoma H460 18, and hepatoma cell line Hep3B 19.

In this report, after treatment with PD0166285, B16 cells were also arrested in the early G1 phase within at least 4 hours. Here we indicate novel antiproliferative activities to block adhesion of the Wee1 inhibitor PD0166285 having shown microtubule destabilization, and depression of cyclin D expression.

Methods

Tumor 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 counting

Cell proliferation was analyzed by counting the cells. B16 cells (1 × 10⁵cells 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.

Drug

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

Antibodies

The present study used: antibodies against cyclin D, cyclin, p16, Wee1, Rb, and anti-α-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA), and against p21 and p27 (Transduction Laboratories, San Diego, CA). Phospho-Plus cdc2 (Tyr15) antibody kit was obtained from New England BioLab (Beverly, MA).

Flow Cytometry and Cell Cycle Analysis

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).

Morphologic observation by immunofluorescence confocal microscopy

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.

Immunoblotting analysis

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.

Total RNA extraction, cDNA synthesis, and reverse transcription (RT)-polymerase chain reaction (PCR)

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).

SYBR Green real-time quantitative PCR

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.

Results

PD0166285 suppressed cell proliferation by inducing G0/1 cell cycle arrest

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.

Figure 1

Effects of PD0166285 treatment on the suppression of cell proliferation and the cell cycle distribution

Effects of PD0166285 treatment on the suppression of cell proliferation and the cell cycle distribution. A: The cells were harvested after treatment with (0, 0.5, 1.0, or 2.0 μM) PD0166285 (including DMSO vehicle) for the indicated times, and then the cells were counted. B: The cells were harvested after treatment with 0.5 μM for the indicated period, fixed, stained with PI, and analyzed for DNA content by flow cytometry. The percent of cells in the cell cycle after treatment were indicated on lower part of the flow cytometry graphs.

PD0166285 inhibited microtubule stabilization

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).

Figure 2

Effects of PD0166285 treatment on the microtubule stabilization

Effects of PD0166285 treatment on the microtubule stabilization. A: Cells were fixed and stained with PI (red) and anti-α-tubulin antibody followed by FITC-labeled secondary antibody (green). Shown is a typical image under laser confocal microscopy. B: magnifications at 0 h, 2 h, and 4 h. Bar: 10 μm

PD0166285 inhibited Wee1 activity and progressed from the G2 to G1 phase

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)

Figure 3

Effects of PD0166285 on the G2 phase

Effects of PD0166285 on the G2 phase. A: Immunoblot analysis of anti-cdc2, anti-phosphorylated cdc2-tyr15, and Wee1 was performed in lysates of with 0.5 μM PD0166285 treated for 0 h, 2 h, or 4 h. B: Subcellular location of cyclin B was observed. After 4 h of treatment with 0.5 μM PD0166285, cells were fixed and doubly stained for immunofluorescence with PI (red) and anti-cyclin B1 (green). Shown are typical merged images obtained by confocal microscopy. Bar: 10 μm.

Localization of Wee1 showed expression upon cell adhesion

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.

Figure 4

Effects of PD0166285 on the cellular location of Wee1 and adhesion

Effects of PD0166285 on the cellular location of Wee1 and adhesion. A: After 4 h of PD0166285 treatment, cells were fixed and doubly stained for immunofluorescence with PI (red) and anti-Wee1 (green). B: After 4 h of 0.5 μM PD0166285 treatment, morphologic changes and double stained with anti-α-tubulin (green) and anti-Wee1 (red) were observed. Shown are typical images obtained by laser confocal microscopy. Bar: 10 μm.

Cell cycle-related proteins were altered

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).

Figure 5

Effects of PD0166285 on expression related protein of cell cycle regulators

Effects of PD0166285 on expression related protein of cell cycle regulators. A: Lysates (30 μg) were prepared from cells after treatment with 0.5 μM PD0166285 for the indicated period (0 h, 2 h and, 4 h) were subjected to immunoblot analysis with anti-cyclin D, anti-cyclin E, anti-p16, anti-p21, and anti-p27 antibodies. B: Cell lysates after treatment with 0.5 μM PD0166285 for long periods treatment; (0 h, 4 h, and 24 h) were subjected to an immunoblot analysis with Anti-Rb antibody and cell cycle suppression directly molecule p21 and p27.

PD0166285 repressed cyclin D transcription

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.

Figure 6

Effects of PD0166285 on cyclin D transcription

Effects of PD0166285 on cyclin D transcription. A: RT-PCR analysis of cyclin D mRNA in 0.5 μM PD0166285 treated cells. RT yielded a cDNA template for PCR amplification with specific-specific primers. GAPDH expression was analyzed as a control. B: Quantitative real-time PCR analysis of specific mRNA after treatment with 0.5 μM PD0166285. Three independent experiments were carried out.

Discussion

We studied the mechanisms by which the Wee1 inhibitor PD0166285 suppressed growth of the B16 mouse melanoma cell line. In previous reports, G2 checkpoint abrogation by 0.5 μM of PD0166285 was demonstrated to kill cancer cells 1821. Deregulated cell cycle progression ultimately may have damaged the cancer cells. When those authors examined toxicity, the highest dose of PD0166285 was 0.5 μM for exposure periods no more than 6 hours in some cell lines. When we exposed the hepatoma carcinoma cell line Hep3B to PD0166285, after 4 hours of treatment the Hep3B cells entered G1 arrest and after 6 hours the cells floated in the medium 19. B16 melanoma cell line cells entered G1 arrested after 4 hours and the cells floated after 24 hours at 0.5 μM PD0166285. B16 cells showed an arrested cell proliferation for 4 hours, but no apoptosis was not observed after 24 hours of treatment by 0.5 μM PD0166285. After 24 hours, the cell were observed to be floating and dead (Figure 1A).

PD0166285 is the safe reagent. At least, 0.3 μM UCN-1 and 3 mM caffeine can induce the dephosphorylation of cdc2-Ty15. PD0166285 can induce the dephosphorylation at 0.5 μM which is very small concentrations. Moreover, for an in vivo assay, we injected 500 μl of 20 μM PD0166285 into the peritoneum cavity of B6 mice for 4 weeks. However the injected reagent was no negative effects to these mice (Data not shown). 0.5 μM PD0166285 might be therefore reagent with the peculiarity and safe for normal cells.

We could not investigate a human melanoma cell line and other cell lines, but such cell arrest and cell death could be observed in some cell lines including a human hepatoma cell line in the previous papers1819. 0.5 μM PD0166285 thus appears to induce cell arrest in many cancer cell lines.

We found that PD0166285 rapidly brought about progression to cell cycle arrest in early G1 after a cell division phase. At a concentration of 0.5 μM, PD0166285 inhibited cdc2 inaction by phosphorylation at Ty15 (Figure 3A). It is reported that kinase activity of Wee1 is regulated by self-phosphorylation. At 4 hours, expression of Wee1 showed little change. Cyclin B protein expression was not seen by immunofluorescence microscopy (Figure 3B). B16 cells arrested in the early G1 phase at 4 hours, according to flow cytmetry (Figure 1). But cell death was not seen until 4 hours of exposure to PD0166285 at 0.5 μM.

These findings indicate that the effect of 0.5 μM PD0166285 for G1 cell arrest is Wee1 activity in an unusual way. The Wee1 knock down (siRNA experiment) might thus help to elucidate the direct or non-specific response. However, further investigation is called for.

The most surprising observation was made after 4 hours of treatment with 0.5 μM PD0166285. Observed by immunofluorescence microscopy, microtubule polymerization failed to occur in B16 cells treated with PD0166285. To clarify these observations we carried out immunofluorescence microscopy (Figure 2B), and flow cytometry (data not shown) after 2 hours of exposure. The cells in resting phase showed no cell cycle progression, and no clearly evident abnormalities. However, microtubule polymerization was blocked in all cells after 4 hours of PD0166285 treatment. Cyclin D was suppressed at 4 hours. PD0166285 treatment for 4 hours suppressed cyclin D protein expression and transcription. These observations indicated that PD0166285 arrested the cell cycle in the early G1 phase, accompanied by inhibition of microtubule stabilization and suppression of cyclin D expression by imperfect adhesion.

Changes in cell architecture are required for progression through the cell cycle. The most dramatic of these is a complete recognition of the cytoskeleton triggered by proteins phosphorylated by cyclinB-cdc2 such as the kinesin-like motor proteins required for proper assembly of mitotic spindle 2223, as well as the actin binding protein caldesmon and several regulators of small GTPases, Rho and CDC42, that mediate rearrangements of the actin cytoskeleton during mitosis 242526. Recent data have indicated that plasma membrane lipid rafts are involved in signal transduction events; raft formation is initiated by cell adhesion to the extracelluler matrix, which is mediated by transmembrane proteins called integrins 272829. Lipid rafts are directed by integrins to target Rho and Rac GTPases. Microtubule stabilization is regulated by Rho-mDia signaling and Rac-PAK signaling 30, which therefore occur downstream of integrin-mediated cell adhesion via lipid raft formation. Focal adhesion kinase (FAK) has been described as a critical mediator of integrin signaling, allowing FAK to influence a variety of processes, including the broadening of the cell attachment, migration, survival and cycle progression.

Cyclin D expression begins to become evident after cell adhesion. Two pathways separately act on cyclin D expression at the early G1 and middle G1 phase. Early in G1, integrin-Rac is important for cyclin D expression when cell cycling is initiated after cell adhesion. Rac was reported to stimulate the expression of CCND1 mRNA in both fibroblasts and epithelial cells 31. NF-κB has been implicated as Rac effectors 3233. Although the relationship between reactive oxygen species and cyclin D1 has yet to be elucidated, human and rodent CCND1 promoters contain functional NF-κB binding sites. In the mid G1 phase, some hours after the early G1 phase, several studies have shown that sustained signaling from extracelluler signal regulated kinases (ERK) stimulates the production of CCND1 mRNA in mesenchymal cells. Integrin signaling, which had originally induced cyclin D expression in early G1, is suppressed in the mid G1 phase 34. Instead, various growth factors act via ERK to up-regulate cyclin D expression in mid G1 35.

PD0166285 inhibited microtubule polymerization while suppressing cyclin D expression. Finally, Rb demonstrated inactive by dephosphorylation (Figure 5B), and the cell cycle was completely arrested in early G1 36. Importantly, adhesion-related molecules such as integrin, Rho, Rac, and FAK are highly complex and involve considerable cross talk in signaling 37. Rho, Rac, and FAK activity were not analyzed in this study, but will be investigated in the future.

Microtubule stabilization failure might involve Wee1 inactivation or a direct drug effect of PD0166285 on microtubules. The role of Wee1 is not completely understood. Our result suggested there was Wee1 colocalization with α-tubulin (Figure 4B), as has been suggested previously 38. Wee1 might affect tubulin directly, while other reports have indicated a relationship between Wee1 and the Src homologue protein Crk, which is associated with FAK signaling 3940. Recently, a novel Wee1 inhibitor related to compounds showing Src kinase homology was reported to arrest the cell cycle in the S phase41. Wee1 might act upon FAK signaling with respect to signal transmission involving a Src-homology protein. As a result, Wee1 may be more than a G2 checkpoint nullifier, since it also affects cell adhesion molecules.

Conclusion

In this study we demonstrated a novel cell cycle-regulating effect of the Wee1 inhibitor PD0166285, a pyrido (2,3-d) pyimidine, in B16 mouse melanoma cells. PD0166285 inhibited Wee1 function, abrogated the G2 checkpoint, and induced early cell division. PD0166285 blocked microtubule stabilization, and inhibited cyclin D expression. These effects thus suggest that Wee1 inhibitor may thus be a potentially effective anti-cancer drug.

Competing interests

The author(s) declare that they have no competing interests.

Authors' contributions

O.H. designed and supervised the study and prepared the manuscript. M.S Performed cell culture and molecular experiments include western blot, Microscopy, etc. T.N performed real-time PCR. K.S assisted western blot. M.S, H.K, and T.T advised and assisted molecular experiments and analysis of cellular change by microscopy. T.U and M.S participated in design of the study. All authors read and approved the final manuscript.

Acknowledgements

This study was supported by grants from the Ministry of Education, Science, Sports, and Culture of Japan as part of a project for establishing new high technology research centers (the 21th century COE program)

Cancer cell cycles Sherr CJ Science 1996 274 1672 1677 10.1126/science.274.5293.1672 8939849 G2 checkpoint abrogators as anticancer drugs Kawabe T Mol Cancer Ther 2004 3 513 519 15078995 Universal control mechanism regulating onset of M-phase Nurse P Nature 1990 344 503 508 10.1038/344503a0 2138713 Checkpoint pathways come of age. Nurse P Cell 1997 91 865 867 10.1016/S0092-8674(00)80476-6 9428508 Regulation of the human WEE1Hu CDK tyrosine 15-kinase during the cell cycle Watanabe N Broome M Hunter T EMBO J 1995 14 1878 1891 398287 7743995 The role of Cdc2 feedback loop control in the DNA damage checkpoint in mammalian cells. Poon RY Chau MS Yamashita K Hunter T Cancer Res 1997 57 15;57(22):5168-78. 5168 5178 9371520 Overproduction of Human Myt1 Kinase Induces a G2 Cell Cycle Delay by Interfering with the Intracellular Trafficking of Cdc2-Cyclin B1 Complexes Liu F Rothblum-oviatt C Ryan CE Piwnica-Worms H Mol Cell Biol 1999 19 5113 5123 84354 10373560 Checkpoints on the road to mitosis. Russell P Trends Biochem Sci 1998 10 399 402 10.1016/S0968-0004(98)01291-2 Inactivation of the p34cdc2-cyclin B complex by the human WEE1 tyrosine kinase. Parker LL Piwnica-Worms H Science 1992 257 1955 1957 10.1126/science.1384126 1384126 Cell cycle regulation of human WEE1 McGowan CH Russell P EMBO J 1995 14 2166 2175 398322 7774574 Coupling of mitosis to the completion of S phase through Cdc34-mediated degradation of Wee1. Michael WM Newport J Science 1998 282 . 1998 Dec 4;282(5395):1886-9 1886 1889 10.1126/science.282.5395.1886 9836638 Control of Swe1p degradation by the morphogenesis checkpoint Sia RAL Bardes ESG Lew DJ EMBO J 1998 17 6678–6688 10.1093/emboj/17.22.6678 Replication checkpoint enforced by kinases Cds1 and Chk1 Boddy MN Furnari, B., Mondesert, O., Russell, P. Science 1998 280 909 912 10.1126/science.280.5365.909 9572736 Chk1 is a wee1 kinase in the G2 DNA damage checkpoint inhibiting cdc2 by Y15 phosphorylation J.O’Connell M M.Raleigh J M.Verkade H Nurse P EMBO J 1997 16 545–554 Timing of cyclin D1 expression within G1 phase is controlled by Rho. Welsh CF Roovers K Villanueva J Liu Y Schwartz MA Assoian RK Nat Cell Biol 2001 3 950 957 10.1038/ncb1101-950 11715015 Expression of Cyclin E Renders Cyclin D-CDK4 Dispensable for Inactivation of the Retinoblastoma Tumor Suppressor Protein, Activation of E2F, and G1-S Phase Progression Keenan SM Lents NH Baldassare JJ J Biol Chem 2004 279 5387–5396 Inhibition of Cdk4 Activity Enhances Translation of p27kip1 in Quiescent Rb-negative Cells Gonza'lez T Seoane M Caamano P Vinuela J Domi´nguez F Zalvide J J Biol Chem 2003 278 12688–12695 Radiosensitization of p53 Mutant cells by PD0166285, a novel G(2) Checkpoint Abrogator Wang Y Li J Booher RN Kraker A Lawrence T Leopold WR Sun Y Cancer RES 2001 61 8211 8217 11719452 Inhibition of proteasome-dependent degradation of Wee1 in G2-arrested Hep3B cells by TGF beta 1. Hashimoto O Ueno T Kimura R Ohtsubo M Nakamura T Koga H Torimura T Uchida S Yamashita K Sata M Mol Carcinog 2003 36 Apr;36(4):171-82. 171 182 10.1002/mc.10111 12669309 Suppression of transforming growth factor-beta results in upregulation of transcription of regeneration factors after chronic liver injury Nakamura T Ueno, T., Sakamoto, M., Sakata, R., Torimura, T., Hashimoto, O., Ueno, H., and Sata, M. J Hepatol 2004 41 974 982 10.1016/j.jhep.2004.08.015 15582131 Wild-type TP53 inhibits G(2)-phase checkpoint abrogation and radiosensitization induced by PD0166285, a WEE1 kinase inhibitor. Li J Wang Y Sun Y Lawrence TS Radiat Res 2002 157 2002 Mar;157(3):322-30. 322 330 10.1667/0033-7587(2002)157[0322:WTTIGP]2.0.CO;2 11839095 Phosphorylation by p34cdc2 regulates spindle association of human Eg5, a kinesin-related motor essential for bipolar spindle formation in vivo. Blangy A Lane, H. A., d'Herin, P., Harper, M., Kress, M., Nigg, E.A. Cell 1995 83 1159 1169 10.1016/0092-8674(95)90142-6 8548803 Targets of the cyclin-dependent kinase Cdk1. Ubersax JA Woodbury, E.L., Quang, P.N., Paraz, M., Blethrow, J.D., Shah, K., Shokat, K.M., Morgan, D.O. Nature 2003 425 859 864 10.1038/nature02062 14574415 Cell migration: integrating signals from front to back. Ridley AJ Schwartz MA Burridge K Firtel RA Ginsberg MH Borisy G Parsons JT Horwitz AR Science 2003 302 . 2003 Dec 5;302(5651):1704-9. 1704 1709 10.1126/science.1092053 14657486 Rho and Rac take center stage Burridge K Wennerberg K Cell 2004 116 167 179 10.1016/S0092-8674(04)00003-0 14744429 Rho GTPases in cell biology. Etienne-Manneville S Hall A Nature 2002 420 629 635 10.1038/nature01148 12478284 Integrins regulate Rac targeting by internalization of membrane domains del Pozo MA Alderson NB Kiosses WB Chiang HH Anderson RG Schwartz MA Science 2004 303 839 842 10.1126/science.1092571 14764880 Localized stabilization of microtubules by integrin- and FAK-facilitated Rho signaling. Palazzo AF Eng CH Schlaepfer DD Marcantonio EE Gundersen GG Science 2004 303 836 839 10.1126/science.1091325 14764879 Integrin Signaling and lipid Rafts del Pozo MA Cell Cycle 2004 6 725 728 Cell biology. Integrins, rafts, Rac, and Rho Guan J Science 2004 303 773 774 10.1126/science.1094376 14764856 Effects of Rho Kinase and Actin Stress Fibers on Sustained Extracellular Signal-Regulated Kinase Activity and Activation of G1 Phase Cyclin-Dependent Kinases Roovers K Assoian RK Mol Cell Biol 2003 23 4283–4294 10.1128/MCB.23.12.4283-4294.2003 Cyclic mechanical strain inhibits skeletal myogenesis through activation of focal adhesion kinase, Rac-1 GTPase, and NF-kappaB transcription factor. Kumar A Murphy R Robinson P Wei L Boriek AM FASEB J 2004 18 2004 Oct;18(13):1524-35. 1524 1535 10.1096/fj.04-2414com 15466361 Rac1 and RhoG promote cell survival by the activation of PI3K and Akt, independently of their ability to stimulate JNK and NF-kappaB. Murga C Zohar, M., Teramoto, H., Gutkind JS Oncogene 2002 21 207 216 10.1038/sj.onc.1205036 11803464 Nuclear translocation of LIM kinase mediates Rho-Rho kinase regulation of cyclin D1 expression. Roovers K Klein EA Castagnino P Assoian RK Dev Cell 2003 5 273 284 10.1016/S1534-5807(03)00206-5 12919678 Characterization of a Rac1 Signaling Pathway to Cyclin D1 Expression in Airway Smooth Muscle Cells Page K Li J Hodge JA Liu PT Hoek TLV Becker LB Pestel RG Rosneri MR Hershenson MB J Biol Chem 1999 274 22065–22071 Cyclin Dl/Cdk4 Regulates Retinoblastoma Proteinmediated Cell Cycle Arrest by Site-specific Phosphorylation Connell-Crowley L Harper JW Goodrich"t DW Mol Biol Cell 1997 8 Vol. 8, 287-301, February 1997 287 301 276080 9190208 Networks and crosstalk: integrin signalling spreads. Schwartz MA Ginsberg MH Nat Cell Biol 2002 4 E65 68 10.1038/ncb0402-e65 11944032 Subcellular localisation of human wee1 kinase is regulated during the cell cycle Baldin V Ducommun B J Cell Science 1995 108 2425 2432 7673359 Wee1-regulated Apoptosis Mediated by the Crk Adaptor Protein in Xenopus Egg Extracts Smith JJ Evans EK Murakami M Moyer MB M. Arthur Moseley George Vande Woude Kornbluth S J Cell Biol 2000 151 1391–1400 10.1083/jcb.151.7.1391 Apoptotic Regulation by the Crk Adapter Protein Mediated by Interactions with Wee1 and Crm1/Exportin Smith JJ Richardson DA Kopf J Minoru Yoshida Hollingsworth RE Kornbluth S Mol Cell Biol 2002 22 1412–1423 S-phase Inhibition of Cell Cycle Progression by a Novel Class of Pyridopyrimidine Tyrosine Kinase Inhibitors Mizenina OA Moasser MM Cell Cycle 2004 3 6 790 803 15118413

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