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Genes to Cells (2005) 10, 655-663. doi:10.1111/j.1365-2443.2005.00866.x
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Activation of c-Jun amino-terminal kinase by GDNF induces G2/M cell cycle delay linked with actin reorganization

Toshifumi Fukuda1,{dagger}, Naoya Asai1, Atsushi Enomoto1 and Masahide Takahashi1,2,*

1 Department of Pathology, and 2 Division of Molecular Pathology, Center for Neurological Disease and Cancer, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, 466-8550, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
It is well known that the cell cycle is controlled by several cyclin/cyclin-dependent kinase (Cdk) complexes whose expression and phosphorylation states vary with orderly periodicity. During the cell cycle, activity of the cyclin/Cdk complexes can be regulated directly or indirectly by a number of molecules, including protein kinases and phosphatases, p53, and Cdk inhibitors. Here, we show that the addition of glial cell line-derived neurotrophic factor (GDNF) induced G2/M cell cycle delay in human SK-N-MC neuroectodermal tumor cells that express RET tyrosine kinase, accompanying actin reorganization. Cell cycle delay at G2/M was characterized by accelerated and prolonged Cdc2 phosphorylation and stabilization of cyclin B1 and Wee1 kinase expression. Interestingly, we found that phosphorylation and/or expression of Cdc2, cyclinB1, and Wee1 was controlled by the Rac1/c-Jun NH2-terminal kinase (JNK) pathway. Immunohistochemical analysis suggested that the G2/M cell cycle delay may be necessary to prevent the mitotic progression of SK-N-MC cells with perturbed actin cytoskeletons.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
A variety of extracellular stimuli, such as growth factors and cytokines, are required for differentiation and determination of cell fate during normal development. A relationship between cellular differentiation and cell cycle exit has been shown in many systems, such that the differentiation machinery appears to affect the cell cycle (Edlund & Jessell 1999). For example, nerve growth factor (NGF) can induce differentiation, such as axon-like protrusive elongation, in PC12 cells with accompanying decreases in Cdc2 and Cdk2 activity (Dobashi et al. 2000). Interestingly, inhibition of Cdc2 and Cdk2 activities alone can also induce neuronal differentiation in the absence of NGF, which indicates that cell cycle molecules may be involved in the regulation of neuronal cell differentiation. Thus, it is possible that the expression of neurotrophic factors in spatially and temporally appropriate manners is required for the normal development of the nervous system through the regulation of the cell cycle.

Glial cell line-derived neurotrophic factor (GDNF) and related molecules neurturin, artemin, and persephin, play crucial roles in the survival and differentiation of various neurons, including peripheral enteric, sensory, and sympathetic neurons, as well as central motor and dopaminergic neurons (Maniéet al. 2001; Takahashi 2001; Airaksinen & Saarma 2002). These neurotrophic factors signal through multisubunit receptor complexes that contain RET receptor tyrosine kinase and a glycosylphosphatidylinositol-anchored co-receptor called GDNF family receptor {alpha}1-4 (GFR{alpha}1-4) (Jing et al. 1996; Treanor et al. 1996; Klein et al. 1997; Baloh et al. 1998; Enokido et al. 1998). In particular, the GDNF/GFR {alpha}1/RET signaling pathway plays a crucial role in the development of the enteric nervous system and the kidney (Schuchardt et al. 1994; Jijiwa et al. 2004). It has been shown that in response to GDNF stimulation, RET can activate a variety of intracellular signaling pathways, including the RAS/ERK, phosphatidylinositol 3-kinase (PI3K)/AKT, p38 mitogen activated protein kinase (p38MAPK), c-Jun NH2-terminal kinase (JNK), and phospholipase C {gamma} pathways (Asai et al. 1996; Borrello et al. 1996; Chiariello et al. 1998; Besset et al. 2000; Hayashi et al. 2000; Segouffin-Cariou & Billaud 2000; Iwashita et al. 2001; Kurokawa et al. 2001; Mellilo et al. 2001; Murakami et al. 2002). However, it remains to be elucidated whether the GDNF/RET signaling pathway is involved in regulation of the cell cycle.

In addition to the signaling pathways mentioned above, GDNF stimulation can also induce Rac1-guanine nucleotide exchange factor (GEF) activation in SK-N-MC human neuroectodermal tumor cells transfected with the RET gene (designated MC(RET) cells) which leads to lamellipodia formation as a result of actin rearrangement (Fukuda et al. 2002; Maeda et al. 2004). We have previously shown that phosphorylation of a serine residue at amino acid position 696 in RET via a cAMP-dependent mechanism was required for Rac1-GEF activation and lamellipodia formation (Fukuda et al. 2002). When serine 696 was replaced with alanine (S696A mutation), GDNF-mediated Rac1-GEF activity and lamellipodia formation were almost completely abolished in SK-N-MC cells transfected with mutant RET (S696A cells).

In the present study, we compared the effects of GDNF on cell growth and intracellular signaling pathways between MC(RET) and S696A cells. Biological and biochemical analyses revealed that continuous GDNF stimulation induced cell cycle delay at G2/M in MC(RET) cells, but not in S696A cells, which was due to accelerated and prolonged phosphorylation of Cdc2 whose dephosphorylation is essential for mitotic progression. We also showed that the Rac1/JNK pathway was required for this cell cycle delay.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Growth arrest of RET-transfected SK-N-MC cells by GDNF

To investigate the role of GDNF in cell growth, the human primitive neuroectodermal tumor cell line SK-N-MC was transfected with wild-type RET proto-oncogene (MC(RET) cells) and cultured in the absence or presence of GDNF. GDNF treatment caused almost complete growth arrest in two independent transfectants, whereas wild-type SK-N-MC cells showed no growth arrest in response to GDNF (Fig. 1A,B). GDNF-mediated growth arrest was not observed in SK-N-MC cells transfected with mutant RET in which serine 696 was replaced with alanine (S696A cells) (Fig. 1C). As previously reported (Fukuda et al. 2002), the S696 residue in RET is phosphorylated in a cAMP-dependent manner and its phosphorylation is crucial for Rac1 activation and lamellipodia formation in MC(RET) cells. Thus, our findings suggested that Rac1 activity may be involved in MC(RET) cell growth arrest.



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  		Figure 1  Growth arrest of MC(RET) cells by GDNF. (A) SK-N-MC, (B) MC(RET) and (C) S696A cells were grown in the absence or presence of GDNF (100 ng/mL) and cell proliferation assays performed using a cell counting kit (DOJINDO).

 
The S696A mutation impairs the Rac1/JNK signaling pathway

We next investigated the activation of downstream signaling molecules in GDNF-treated transfectants. While similar levels of ERK1/2, AKT, and p38MAPK activation were observed between MC(RET) and S696A cells, JNK was activated in MC(RET) but not in S696A cells (Fig. 2A). Consistent with this finding, c-Jun was activated by GDNF only in MC(RET) cells, with this activation almost completely abolished by treatment with the JNK inhibitor SP600125 (Fig. 2B). Moreover, when dominant-active Rac1 was transiently transfected into S696A cells, JNK activation was induced (Fig. 2C), which indicated that Rac1 activation was required for JNK activation and that the Rac1/JNK pathway was specifically impaired in S696A cells. On the other hand, expression of dominant-active Rac1 could not activate Akt, and p38MAPK in S696A cells (Fig. 2C).



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  		Figure 2  The S696A mutation specifically impairs the Rac/JNK pathway. (A) Impaired JNK activation in S696A cells. MC(RET) and S696A cells were treated with GDNF (100 ng/mL) for the indicated times and prepared lysates (20 µg) analyzed by Western blotting with the designated antibodies. A band detected by anti-phosphotyrosine (pY) antibody represents the 170 kDa phosphorylated RET protein. IB: immunoblotting. (B) c-Jun activation in GDNF-treated MC(RET) cells. MC(RET) and S696A cells were cultured in the presence or absence of the JNK inhibitor SP600125 (20 µM) (Bennett et al. 2001) for 1 h, followed by GDNF stimulation for 20 min. Cell lysates were analyzed by Western blotting with anti-phospho-c-Jun or anti-c-Jun antibodies. (C) JNK activation by dominant active Rac1. An expression plasmid carrying dominant active Rac1 (Rac1 DA) was transiently transfected into S696A cells and incubated for 48 h. AKT, p38MAPK and JNK activation was analyzed using anti-phospho-AKT, phospho-p38MAPK and phospho-JNK antibodies.

 
GDNF induces JNK-dependent cell cycle delay at G2/M

MC(RET) cells were synchronized at the G1/S transition phase in the presence of 2.5 mM thymidine for 24 h, released in the absence or presence of GDNF, and then analyzed by flow cytometry. Approximately 60% of both GDNF-untreated and -treated cells had reached the G2/M phase 12 h after release (Fig. 3A). Interestingly, 33% of GDNF-untreated MC(RET) cells had re-entered the G1 phase 18 h after stimulation, whereas only 9% of GDNF-treated cells were in G1 at the same time point, which implied that the cell cycle of GDNF-treated cells was delayed at the G2/M phase. On the other hand, both GDNF-untreated and -treated S696A cells showed normal cell cycle progression (Fig. 3B).



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  		Figure 3  GDNF-mediated G2/M cell cycle delay. (A) MC(RET) and (B) S696A cells were synchronized at the G1/S transition phase by 2.5 mM thymidine treatment for 24 h and released in the presence or absence of GDNF (100 ng/mL). Cells were harvested at the indicated times and analyzed by flow cytometry. (C) Similarly, MC(RET) cells were analyzed in the presence of both GDNF and SP600125 (20 µM).

 
To investigate whether JNK is involved in the cell cycle delay observed in MC(RET) cells, the experiments were carried out in the absence or presence of SP600125. The cell cycle progression of GDNF-treated MC(RET) cells were recovered by the addition of SP600125 (Fig. 3C), indicating that the cell cycle delay was dependent on the Rac1/JNK pathway.

Sustained Cdc2 phosphorylation and cyclin B1 and Wee1 kinase expression in GDNF-treated MC(RET) cells

We then investigated the expression and phosphorylation of components of the cell cycle machinery that control the G2/M phase. Cell lysates from synchronized MC(RET) and S696A cells released in the absence or presence of GDNF were analyzed by Western blotting with antibodies against known cell cycle molecules. As shown in Fig. 4A,B, phosphorylation of Cdc2, known to play a crucial role in the G2/M transition, was accelerated and prolonged in GDNF-treated MC(RET) cells, but not in GDNF-treated S696A cells. High levels of Cdc2 phosphorylation were detected in MC(RET) cells as early as 8 h after GDNF stimulation and continued for at least a further 24 h (Fig. 4B). Similarly, accelerated and sustained expression of cyclin B1 and Wee1 kinase, the latter responsible for Cdc2 phosphorylation, was observed in MC(RET) cells from 8 to 24 h after GDNF stimulation (Fig. 4B). In contrast, Cdc2 dephosphorylation and Wee1 and cyclin B1 degradation occurred in S696A cells 20 h to 24 h after GDNF stimulation, which indicated a normal cell cycle progression pattern (Fig. 4B). However, phosphorylation levels of Cdc25c phosphatase, involved in the phosphorylation status of Cdc2, were not significantly different between MC(RET) and S696A cells (data not shown). These results suggested that the G2/M cell cycle delay observed in GDNF-treated MC(RET) cells was due to the sustained Cdc2 phosphorylation and cyclin B1 and Wee1 kinase expression.



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  		Figure 4  JNK-dependent G2/M cell cycle delay by GDNF. (A) Accelerated and prolonged Cdc2 phosphorylation by GDNF in MC(RET) cells. Synchronized MC(RET) and S696A cells were released in the absence or presence of GDNF and harvested at the indicated times. Cell lysates (20 µg) were analyzed by Western blotting with anti-phospho-Cdc2 or anti-Cdc2 antibodies. (B) Accelerated and sustained Wee1 kinase and cyclin B1 expression by GDNF. Lysates from synchronized cells were analyzed by Western blotting with the designated antibodies. (C) Inhibition of accelerated Cdc2 phosphorylation by a JNK inhibitor. Synchronized MC(RET) cells were released in the presence of inhibitors, as shown, for 1 h and then incubated with GDNF plus each inhibitor for the indicated times. Cell lysates were analyzed by Western blotting with anti-phospho-Cdc2 or anti-Cdc2 antibodies. SP600125, a JNK inhibitor; PD98059, a MEK1 inhibitor; LY294002, a PI3K inhibitor; SB202190, a p38MAPK inhibitor. (D) Inhibition of accelerated and sustained Wee1 and cyclin B1 expression by SP600125. Lysates from synchronized cells incubated in the presence of GDNF and SP600125 were analyzed by Western blotting with the designated antibodies as described above.

 
To further investigate whether the accelerated and prolonged Cdc2 phosphorylation was dependent on JNK activation, MC(RET) cells were treated with GDNF in the presence of SP600125. Cdc2 phosphorylation in MC(RET) cells at 8 h after GDNF stimulation was almost completely inhibited by SP600125, while treatment with MEK1 inhibitor PD98059, PI3K inhibitor LY294002, or p38MAPK inhibitor SB202190 had no effect (Fig. 4C). In addition, the accelerated and sustained Wee1 and cyclin B1 expression was significantly decreased in SP600125-treated MC(RET) cells upon GDNF stimulation (Fig. 4D). Thus, our results indicated that in GDNF-treated MC(RET) cells, Cdc2, cyclin B1, and Wee1 cell cycle molecules appeared to be regulated by the JNK pathway.

Association of G2/M delay with actin reorganization

We previously reported that GDNF induced lamellipodia formation in MC(RET) cells following Rac1 activation, but did not affect wild-type SK-N-MC or S696A cells (Fukuda et al. 2002). To examine whether lamellipodia formation was cell cycle phase-dependent and therefore associated with the G2/M delay, GDNF-untreated or-treated MC(RET) cells were stained with FITC-phalloidin and anti-cyclin B1 antibody. After 30 min GDNF-treatment, lamellipodia formation was induced in approximately 30% of cyclin B1-negative MC(RET) cells, but was only rarely observed in cyclin B1-positive cells (Fig. 5A,B), which suggested that lamellipodia were induced in the G1 phase rather than the G2/M phase. The finding that cyclin B1 was detected mainly in the cytoplasm (Fig. 5A) implied that most of the cyclin B1-positive cells had not yet entered mitosis (Hagting et al. 1999; Ohi & Gould 1999). After 24 h GDNF stimulation, lamellipodia formation was detected in about 50% and 10% of cyclin B1-negative and -positive cells, respectively. Interestingly, after 5-days culture in the presence of GDNF, most cells became cyclin B1-positive, with approximately 70% showing lamellipodia formation (Fig. 5A,B). Both cytoplasmic and nuclear cyclin B1 was detected in most MC(RET) cells after 5 days of GDNF treatment (Fig. 5A), which suggested that these cells had been arrested during the early M phase (Hagting et al. 1999; Ohi & Gould 1999). Moreover, a significant population of these GDNF-treated MC(RET) cells (~50%) had undergone apoptosis, as shown by TdT-mediated dUTP-FITC nick-end labeling (TUNEL) analysis (Fig. 5C,D).



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  		Figure 5  Sustained lamellipodia formation and cyclin-B1 expression in GDNF-treated MC(RET) cells. (A) Double staining of actin and cyclin B1. After GDNF stimulation for the indicated times, SK-N-MC and MC(RET) cells were stained with FITC-phalloidin (green) and anti-cyclin B1 antibody (red). Lamellipodia formation is indicated by arrows. (B) Quantitative analysis of lamellipodia formation in cyclin B1-negative and -positive MC(RET) cells. Results represent averages from three independent experiments. (C) Detection of apoptotic cells by TUNEL. After GDNF stimulation for the indicated times, SK-N-MC and MC(RET) cells were stained with TUNEL (green) and TRITC-phalloidin (red). (D) Quantitative analysis of apoptosis of GDNF-treated MC(RET) cells. Results represent averages from three independent experiments.

 
In contrast, the percentage of cyclin B1-positive cells and the subcellular localization of cyclin B1 expression did not significantly change in SK-N-MC (Fig. 5A) and S696A cells (data not shown) before or after GDNF stimulation. Neither SK-N-MC nor S696A cells exhibited lamellipodia formation or underwent apoptosis in response to GDNF treatment. Thus, it appeared that the G2/M checkpoint initiated by GDNF functioned to prevent the abnormal cell division of MC(RET) cells with sustained lamellipodia formation.


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
In this report, we showed that GDNF stimulation induced growth arrest in MC(RET) cells, but not in wild-type SK-N-MC cells. G2/M cell cycle delay was observed when MC(RET) cells synchronized at the G1/S transition phase were released in the presence of GDNF. The Rac1/JNK signaling pathway appeared to be required for this cycle delay as cells expressing the RET S696A mutation showed specifically impaired Rac1/JNK signaling and exhibited normal cell cycle progression. In addition, when SK-N-MC cells expressing RET in which tyrosine 1062 was replaced with phenylalanine (Fukuda et al. 2002), were treated with GDNF, cell cycle delay and JNK activation were not induced (data not shown). Consistent with the finding that the cell cycle delay occurred at G2/M, GDNF stimulation induced accelerated and prolonged Cdc2 phosphorylation as well as earlier expression and stabilization of cyclin B1 and Weel kinase in MC(RET) cells. As expected, these events were not observed in GDNF-treated S696A cells that did not show G2/M delay. It has been well documented that Cdc2 dephosphorylation and cyclin B1 degradation are required for mitotic progression and that the phosphorylation status of Cdc2 is controlled by the balance between the activities of Wee1 kinase and Cdc25 phosphatase (Russell & Nurse 1987; Ohi & Gould 1999; Molinari 2000; Pearce & Humphrey 2001; Mikhailov & Rieder 2002). As the phosphorylation levels of Cdc25 were not significantly different between MC(RET) and S696A cells (data not shown), accelerated and prolonged Cdc2 phosphorylation was probably due to sustained Wee1 kinase expression, which resulted in the accumulation of inactive Cdc2/cyclin B1 complexes and G2/M cell cycle delay (Fig. 6).



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  		Figure 6  Proposed pathway of G2/M cell cycle delay triggered by GDNF.

 
Treatment of MC(RET) cells with the JNK inhibitor SP600125 (Bennett et al. 2001; Weston & Davis 2002) inhibited the accelerated and prolonged Cdc2 phosphorylation and the sustained cyclin B1 and Wee1 kinase expression mediated by GDNF. In contrast, treatment with MEK1, PI3-K or p38MAPK inhibitors did not affect the phosphorylation or expression of these proteins in GDNF-treated MC(RET) cells, which confirmed the importance of the Rac1/JNK pathway in the cell cycle delay. Recently, Wada et al. (2004) reported that the MKK7-JNK-c-Jun pathway coupled stress signaling to G2/M cell-cycle progression and cellular senescence in mkk7–/– mouse embryonic fibroblasts. In contrast, our data showed that activation of the Rac1-JNK pathway induced G2/M cell-cycle delay in SK-N-MC cells, which suggested that the biological response to JNK activation may be different depending on cell type. Involvement of JNK in the cell cycle has been reported in a study using human glioblastoma T98G cells (Potapova et al. 2000). Depletion of JNK1 or JNK2 induced cell growth arrest in the S phase, while JNK-specific anti-sense oligonucleotides increased p21cip1/waf1 expression and inhibited Cdk2-cdc2 activity.

GDNF induced lamellipodia formation in MC(RET) cells, but not in S696A cells, as a result of actin rearrangement. Also, Rac1 activation via cAMP-dependent S696 phosphorylation was found to be essential for lamellipodia formation in MC(RET) cells (Fukuda et al. 2002). To investigate the relationship between lamellipodia formation and cell cycle delay, we stained GDNF-treated SK-N-MC and MC(RET) cells with FITC-phalloidin and anti-cyclin B1 antibody. It is known that cyclin B1 accumulates in the late G2 phase and degrades during the M phase. In addition, cyclin B1 is localized mainly in the cytoplasm during G2 and rapidly moves into the nucleus as cells enter mitosis (Hagting et al. 1999; Ohi & Gould 1999). Immunostaining showed that lamellipodia formation was induced mostly in cyclin B1-negative cells 30 min after GDNF stimulation, which suggested that lamellipodia induction occurred in G1 phase cells. Lamellipodia formation was still observed in cyclin B1-negative cells 24 h after GDNF stimulation, although by this stage it also became detectable in approximately 10% of nuclear cyclin B1-positive cells. This meant that some cells exhibiting lamellipodia formation had entered mitosis. Intriguingly, most of the GDNF-treated MC(RET) cells became nuclear cyclin B1-positive 5 days after stimulation, with 70% of such cells showing lamellipodia formation. However, it appeared that these cells could not complete mitosis, as approximately 50% of the cells were TUNNEL-positive. Thus, our findings suggested that MC(RET) cells with lamellipodia formation continuously stimulated with GDNF first arrested in G2/M and then later underwent apoptosis.

Neurotrophic factors, including GDNF and NGF, can induce neuronal differentiation via activation of a variety of intracellular signaling pathways (Fariñas 1999; Kaplan & Miller 2000; Maniéet al. 2001; Patapoutian & Reichardt 2001; Takahashi 2001; Airaksinen & Saarma 2002; Heerssen & Segal 2002). In particular, lamellipodia formation induced by Rac1 activity is known to be critical for neuritegenesis (Mueller 1999; Luo 2000; Dickson 2001). However, it is not yet known whether neurotrophic factors regulate the cell cycle that affects neuronal differentiation (Ohnuma et al. 2001). Neuronal differentiation can occur after a certain number of divisions of precursor cells at specific developmental stages, with actin reorganization, such as lamellipodia formation, induced following the final cell cycle. Thus, it is possible that the observed G2/M checkpoint may function to prevent abnormal division of neuronal cells, with actin rearrangement mediated by neurotrophic factors, and may play a role in the normal development of the nervous system.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Cell lines and transfection

SK-N-MC primitive neuroectodermal tumor cells were transfected with the human RET gene with or without the S696A mutation as previously described (Fukuda et al. 2002). Briefly, human RET cDNA was inserted into the pcDNA3.1/Zeo plasmid vector (Invitrogen) and point mutations introduced using a QuikChange Site-Directed Mutagenesis Kit (Stratagene). Transfections were performed by the calcium phosphate precipitation method using a Mammalian Transfection Kit (Stratagene). To obtain stable transfectants, colonies were selected in the presence of zeocin (150 µg/mL; Invitrogen). To synchronize cells at the G1/S transition phase of the cell cycle, cells were treated with 2.5 mM thymidine for 24 h, rinsed twice with PBS and released in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum.

Cell proliferation assay

Cells were seeded at a density of 1 x 103 cells per well in 96-well microtiter plates and grown in the absence or presence of GDNF (100 ng/mL). At each time point, WST-1 (cell counting kit, DOJINDO, Kumamoto, Japan) was added to each well and incubated at 37 °C for 3 h, and the absorbance of the supernatant at 450 nm and 620 nm measured. All results were based on at least five parallel measurements at each time point and were calculated as the averages of three independent experiments.

Antibodies

Anti-RET antibody was developed as previously described (Kurokawa et al. 2001). Anti-phosphotyrosine was purchased from Upstate Biotechnology Inc. Anti-phospho-JNK, anti-phospho-p38MAPK, anti-phospho-Cdc2, anti-phospho-cJun, anti-phospho-Cdc25c, anti-phospho-ERK and anti-phospho-AKT polyclonal antibodies were purchased from New England BioLabs. Anti-Wee1, anti-JNK and anti-cyclin B1 polyclonal antibodies were purchased from Santa Cruz Biotechnology.

Western blotting

Cells untreated or treated with GDNF (100 ng/mL) were lysed in sodium dodecyl sulfate (SDS) sample buffer (20 mM Tris-HCl, pH 6.8, 2 mM EDTA, 2% SDS, 10% sucrose, 20 µg/mL bromophenol blue (BPB), 80 mM dithiothreitol (DTT)). After boiling for 3 min, equal amounts of protein were subjected to SDS-8% polyacrylamide gel electrophoresis and transferred to polyvinylidine difluoride (PVDF) membranes (Millipore). Membranes were blocked for 30 min at room temperature in 3% albumin in TPBS (phosphate buffered saline containing 0.5% Tween 20) with gentle shaking and incubated with primary antibodies overnight at 4 °C. After washing three times in TPBS, membranes were incubated with secondary antibody conjugated to horseradish peroxidase (swine anti-rabbit IgG-HRP, Dako) for 1 h at room temperature. Reactions were assessed using an enhanced chemiluminescence detection kit (ECL, Amersham Pharmacia Biotech) according to the manufacturer's instructions.

Immunofluorescence

Cells were fixed in PBS containing 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100. After incubation in PBS containing 1% bovine serum albumin (BSA) for 15 min, cells were reacted with anti-cyclin B1 polyclonal antibody for 1 h at room temperature and then incubated with tetramethylrhodamine-5-isocyanate (TRITC)-conjugated anti-rabbit IgG antibody and FITC-phalloidin. For TUNEL assay, the cells were reacted with FITC-labeled dUTP and terminal transferase (TaKaRa), followed by incubation with TRITC-conjugated phalloidin. Staining was analyzed using a confocal microscope (Bio-Rad).

Flow cytometry

Cell cycle profiles were analyzed by staining intracellular DNA with propidium iodide followed by flow cytometry using a FACS-Calibur (Becton-Dickinson).


   Acknowledgements
 
We thank K. Kaibuchi and M. Fukata for the dominant-active Rac1 plasmid, and H. Okayama for the human Wee1 plasmid. This work was supported by Grants-in-Aid for Center of Excellence (COE) Research, Scientific Research (A), and Scientific Research Priority Areas ‘Cancer’ from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by a Grant from the Uehara Memorial Foundation.


   Footnotes
 
Communicated by: Kozo Kaibuchi

{dagger}Present address: Department of Developmental Neurobiology, Institute of Development, Aging and Cancer, Tohoku University, 4-1, Seiryo, Aoba, Sendai 980-8575, Japan. Back

* Correspondence: E-mail: mtakaha{at}med.nagoya-u.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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Received: 18 February 2005
Accepted: 29 March 2005




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