M phase of the cell cycle

The International Journal of Biochemistry & Cell Biology 45 (2013) 1042–1050

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Role of polyamines at the G1 /S boundary and G2 /M phase of the cell cycle Tomoko Yamashita a,1 , Kazuhiro Nishimura a,1 , Ryotaro Saiki a,b , Hiroyuki Okudaira a , Mayuko Tome a , Kyohei Higashi a , Mizuho Nakamura c , Yusuke Terui c , Kunio Fujiwara d , Keiko Kashiwagi c , Kazuei Igarashi a,b,∗ a

Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan Amine Pharma Research Institute, Innovation Plaza at Chiba University, 1-8-15 Inohana, Chuo-ku, Chiba 260-0856, Japan c Faculty of Pharmacy, Chiba Institute of Science, 15-8 Shiomi-cho, Choshi, Chiba 288-0025, Japan d Faculty of Biotechnology and Life Science, Sojo University, 4-22-1 Ikeda, Kumamoto 860-0082, Japan b

a r t i c l e

i n f o

Article history: Received 26 November 2012 Received in revised form 2 February 2013 Accepted 24 February 2013 Available online 14 March 2013 Keywords: Polyamine deficiency Cell cycle p27Kip1 Translational regulation Cytokinesis

a b s t r a c t The role of polyamines at the G1 /S boundary and in the G2 /M phase of the cell cycle was studied using synchronized HeLa cells treated with thymidine or with thymidine and aphidicolin. Synchronized cells were cultured in the absence or presence of ␣-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, plus ethylglyoxal bis(guanylhydrazone) (EGBG), an inhibitor of S-adenosylmethionine decarboxylase. When polyamine content was reduced by treatment with DFMO and EGBG, the transition from G1 to S phase was delayed. In parallel, the level of p27Kip1 was greatly increased, so its mechanism was studied in detail. Synthesis of p27Kip1 was stimulated at the level of translation by a decrease in polyamine levels, because of the existence of long 5 -untranslated region (5 -UTR) in p27Kip1 mRNA. Similarly, the transition from the G2 /M to the G1 phase was delayed by a reduction in polyamine levels. In parallel, the number of multinucleate cells increased by 3-fold. This was parallel with the inhibition of cytokinesis due to an unusual distribution of actin and ␣-tubulin at the M phase. Since an association of polyamines with chromosomes was not observed by immunofluorescence microscopy at the M phase, polyamines may have only a minor role in structural changes of chromosomes at the M phase. In general, the involvement of polyamines at the G2 /M phase was smaller than that at the G1 /S boundary. © 2013 Elsevier Ltd. All rights reserved.

1. Introduction Polyamines (putrescine, spermidine and spermine) are present at millimolar concentrations in both prokaryotic and eukaryotic cells, and play regulatory roles in cell proliferation and differentiation (Cohen, 1998; Igarashi and Kashiwagi, 2010). Polyamines exist mostly as polyamine–RNA complexes and thus affect translation at various steps (Watanabe et al., 1991). In Escherichia coli, polyamines enhance total protein synthetic activity through stimulation of the assembly of 30S ribosomal subunits (Igarashi and Kashiwagi, 2010). Furthermore, polyamines stimulate synthesis of

Abbreviations: CDK, cyclin-dependent protein kinase; DFMO, ␣difluoromethylornithine; EGBG, ethylglyoxal bis(guanylhydrazone); PBS, phosphate-buffered saline; 5 -UTR, 5 -untranslaed region. ∗ Corresponding author at: Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan. Tel.: +81 43 224 7500; fax: +81 43 379 1050. E-mail addresses: [email protected], [email protected] (K. Igarashi). 1 These authors contributed equally to this work. 1357-2725/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biocel.2013.02.021

several kinds of proteins, mainly transcription factors, at the level of translation. These proteins in turn stimulate the synthesis of several kinds of mRNAs encoding proteins important for cell growth. We termed the genes encoding proteins regulated by polyamines at the translation level a “polyamine modulon” (Igarashi and Kashiwagi, 2010; Yoshida et al., 2004). In mammalian cells, overall protein synthesis was increased in parallel with the increase in polyamines (Igarashi and Morris, 1984; Kakinuma et al., 1988), and polyamine stimulation of protein synthesis occurred at the level of initiation (Ogasawara et al., 1989). In addition, we have recently found that specific kinds of protein synthesis are stimulated by polyamines at the level of translation similarly to E. coli (Nishimura et al., 2009). Thus, polyamine stimulation of protein synthesis contributes to polyamine effects on cell growth. It is established that the level of ornithine decarboxylase, a rate limiting enzyme of polyamine biosynthesis, exhibits two rapid and transient peaks during the cell cycle (Fredlund et al., 1995; Nishimura et al., 2007; Pyronnet et al., 2000). The first peak is observed at the G1 /S boundary, and the second occurs at the G2 /M transition. This suggests that polyamines play important roles at distinct stages of cell cycle. During the shift from G1 to S phase, the

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levels of p27Kip1 and p21Cip1/WAF1 , inhibitors of cyclin-dependent protein kinases CDK2 and CDK4 (Nakayama and Nakayama, 1998), were increased by polyamine deficiency (Choi et al., 2000; Ray et al., 1999). Furthermore, polymerization of actin and ␣-tubulin was enhanced by polyamines (Banan et al., 1998; Oriol-Audit, 1978; Pohjanpelto et al., 1981; Savarin et al., 2010). However, a detailed and systematic study of the effects of polyamines on each phase of the cell cycle has not been carried out. In this report, we studied which stage of the cell cycle is more strongly affected by polyamine deficiency using synchronized HeLa cells and specific inhibitors of polyamine biosynthesis – ␣-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (Mamont et al., 1978), and ethylglyoxal bis(guanylhydrazone) (EGBG), a more specific inhibitor of S-adenosylmethionine decarboxylase than MGBG [methylglyoxal bis(guanylhydrazone)] (Igarashi et al., 1984). We found that translation of p27Kip1 mRNA is enhanced at the G1 phase, and cytokinesis is inhibited through inhibition of polymerization of actin and ␣-tubulin at the M phase by polyamine deficiency.

gentamycin and 5% fetal bovine serum (FBS) at 37 ◦ C in an atmosphere of 5% CO2 . Cells were synchronized at G1 /S boundary by treatment with 2 mM thymidine (Sigma–Aldrich) for 24 h (Heintz et al., 1983). Then, effects of polyamine deficiency on the cell division cycle were tested by culturing cells in new medium in the presence and absence of inhibitors of polyamine biosynthesis (0.5 mM DFMO and 0.1 mM EGBG). Where indicated, 5 ␮g/ml aphidicolin (Sigma–Aldrich) was added to the medium to inhibit DNA synthesis (Heintz et al., 1983). Cell-division cycle was monitored by flow cytometry analysis of cellular DNA stained with propidium iodide (Nacalai Tesque Inc.) after fixation of cells with 70% ethanol using EPICS ELITE (Bechman Coulter Inc.). Data were analyzed by Summit R V3.1 (Cytomation, Inc.).

2. Materials and methods

2.3. Western blotting

2.1. Cell culture and synchronization

Western blot analysis was performed as described previously (Nishimura et al., 2005) using Prot Blot Western blot AP system (Promega). Commercially available antibodies were used for the experiments: actin, cyclin D1, cyclin E, p16INK4a and GAPDH from Santa Cruz, CDK4, p27Kip1 , p21Cip1/WAF1 , CDK2, CDC34, PCNA, Rb,

HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Nissui Pharmaceutical Co.) supplemented with 30 ␮g/ml glutamine, 50 U/ml streptomycin, 100 U/ml penicillin G, 50 ␮g/ml

(A)

0h

24 h

2.2. Measurement of polyamine content Polyamine content was measured essentially according to the method described previously (Igarashi et al., 1986).

12 h 18 h 24 h

36 h

(a) Thymidine (2 mM) (b)

Cell number

(a) None 0h

G1: 77.0%

±Aphidicolin (5 µg/ml)

DFMO (0.5 mM) ±Aphidicolin (5 µg/ml) EGBG (0.1 mM) +Aphidicolin +Aphidicolin

Thymidine (2 mM)

(B)

None

12 h G1: 47.8%

18 h 24 h G1: 73.8% G1: 50.1%

Cell number

G1: 77.0%

1C 2C

12 h G1: 41.3%

1C 2C

18 h 24 h G : 76.7% 1 G1: 56.9%

1C 2C

G2/M: 6.0%

36 h G2/M: 4.8%

+Aphidicolin +Aphidicolin

(b) 0.5 mM DFMO and 0.1 mM EGBG 0h

24 h

1C 2C

24 h G2/M: 15.9%

1C 2C

36 h G2/M: 14.3%

1C 2C

(C)

Polyamine (nmol/mg protein)

Intensity of fluorescence of propidium iodide 10 8 6 4 2 0

0

14 14 12 Spermidine 12 Spermine 10 10 8 8 6 6 4 4 2 2 0 0 0 12 24 36 12 24 36 0 12 24 36 Time (h) Time (h) Time (h)

Putrescine

(a) None (b) 0.5 mM DFMO + 0.1 mM EGBG

Fig. 1. Effect of DFMO and EGBG at G1 /S boundary and G2 /M phase. (A) Schematic of experimental design. HeLa cells were treated with 0.5 mM DFMO and 0.1 mM EGBG after synchronization at G1 phase by 2 mM thymidine. After 12 h, cells were treated with 5 ␮g/ml aphidicolin, where indicated. (B) Cell cycle distribution was analyzed by flow cytometry by staining cells with propidium iodide after synchronization at 0, 12, 18, 24 and 36 h cultured in the presence and absence of DFMO and EGBG. Experiments were repeated three times, and the results were reproducible. (C) Level of polyamines were measured as described in Section 2. Level of polyamines did not change significantly when aphidicolon was added. Level of polyamines is shown as means ± S.E. of triplicate determinations.

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Fig. 2. Level of protein kinases and their regulatory proteins involved in transition of G1 to S phase and G2 /M phase. (A) Schematic of protein kinases and their regulatory proteins involved in cell cycle. (B) The level of these proteins was determined by Western blot analysis. Molecular mass of each protein was shown in parenthesis. Cell lysate was prepared from cells cultured with or without 0.5 mM DFMO and 0.1 mM EGBG. 30 ␮g protein was used for the analysis. Experiments were repeated three times, and the results were reproducible. The level of GAPDH was measured as a control.

cyclin A, CDK1, cyclin B, Chk1, CDC25B, Mad2 and CDC27 from Cell cycle I and II Sampler kit, BD Transduction, p45Skp2 from Zymed Lab., and Pirh2 from Calbiochem. Antibodies against KPC1 and KPC2 were kindly supplied from Dr. K.-i. Nakayama, Kyushu University. 2.4. Analysis of the amount of p27Kip1 and GAPDH mRNAs Total RNA was isolated from 2 × 107 cells by the method of Cathala et al. (1983). The amount of p27Kip1 mRNA was estimated by the reverse transcriptase (RT)-PCR method (Heljasvaara et al., 1997). The 5 -end and 3 -end primers used for p27Kip1 mRNA were 5 -AAACGTGCGAGTGTCTAACGGGA-3 (nt 471–493 from the transcription start site of the p27Kip1 gene) and 5 TCGCTTCCTTATTCCTGCGCATTG-3 (complementary to nt 904–927 from the transcription start site of the p27Kip1 gene). As a control, the amount of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was estimated using the 5 - and 3 -end primers (nt 538–560 and complementary to nt 702–725 from the transcription start site of the GAPDH gene). After 25 cycles of PCR, the products were analyzed by gel electrophoresis on 1.2% agarose. The yield of DNA was proportional to the amount of RNA added. Under standard conditions, 0.5 ␮g of total RNA was used for RT-PCR. 2.5. Identification of phosphorylated form of p27Kip1 by two-dimensional (2D) gel electrophoresis Preparation of samples, first-dimension isoelectric focusing (gel size, 17 cm), and second-dimension electrophoresis (gel size, 20 cm × 18 cm) were performed according to BIO-RAD instruction manual (Ready PrepTM sequential extraction kit, protein IEF cell, 2-D starter kit, and protein IIXi 2-D cell) in PROTEAN® IEF Cell. The amount of protein used for first-dimension isoelectric focusing was 1.5 mg.

2.6. Measurement of p27Kip1 and actin synthesis After 1.5 × 106 cells were synchronized by incubation with 2 mM thymidine for 24 h, cells were cultured in the absence and presence of 0.5 mM DFMO and 0.1 mM EGBG for 24 h. Cells were harvested by centrifugation at 1,000 rpm for 10 min, washed with phosphate-buffered saline (PBS) and then cultured for 1 h with 1 ml methionine-free DMEM containing 5% dialyzed FBS and 10 MBq [35 S]methionine. Labeling of cells with [35 S]methionine was stopped by adding 1 ml DMEM containing 10 mg methionine. After cells were washed with PBS, they were suspended with 0.2 ml of a lysis buffer containing 50 mM Tris–HCl, pH 7.5, 0.5% sodium dodecyl sulfate (SDS), 10 mM dithiothreitol, 50 mM NaF, 0.1 mM Na3 VO4 , 1 mM phenylmethylsulfonyl fluoride and 20 ␮M 6-amino2-naphthyl-4-guanidinobenzoate (FUT-175), an inhibitor of serine protease. After boiling the cell lysate for 5 min, the supernatant was obtained by centrifugation at 3,000 rpm for 10 min. To the supernatant containing 4 × 107 cpm of [35 S]methionine-labeled protein, 2 ␮g of anti-p27Kip1 monoclonal antibody (BD Trans Clone 57) and 0.05 ml of 50% (v/v) protein A-Sepharose were added. After p27Kip1 bound to protein A-Sepharose was extracted with 1% sodium deoxycholate and 1% NP-40, the level of p27Kip1 was estimated by 13.5% SDS-PAGE followed by fluorography. Radioactivity of the labeled p27Kip1 was quantified using a BAS2000 II imaging analyzer (Fuji Film). As a control, actin synthesis was similarly estimated using 2 ␮g of anti-actin goat polyclonal antibody (Santa Cruz). 2.7. Immunocytochemistry HeLa cells (2 × 105 ) were loaded in a dish (3.5 cm diameter) containing 22 mm × 22 mm polylysine-coated cover glass. Cells were fixed by the following treatments: ␣-tubulin and vimentin, incubated with 0.1 ml of PBS containing 4% paraformaldehyde at 37 ◦ C

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Fig. 3. Mechanism of increase in p27Kip1 by polyamine deficiency. (A) The level of p27Kip1 and GAPDH mRNAs was estimated by RT-PCR. (B) The level of phosphorylation of p27Kip1 was determined by 2D gel electrophoresis as described in Section 2. Numbers 1, 2, 3 and 4 indicate isoelectric points 5.8, 5.95, 6.24 and 6.5, respectively. No. 4 represents unphosphorylated p27Kip1 , No. 3 may represent posttranslational modification such as glycosylation other than phosphorylation, and No. 1 and No. 2 are phosphoforms of p27Kip1 (Ciarallo et al., 2002). (C) Stability of p27Kip1 was measured by Western blotting through the inhibition of protein synthesis with 10 ␮g/ml cycloheximide. After synchronization with 2 mM thymidine, HeLa cells were cultured in the presence and absence of DFMO and EGBG for 24 h, and cycloheximide was added. Then, the level of p27Kip1 was determined at the designated time of incubation. (D) Level of E3 proteins which are involved in the degradation of p27Kip1 was measured using 30 ␮g protein. Molecular mass of each protein was shown in parenthesis. The level of GAPDH was measured as a control. (E) Synthesis of p27Kip1 and actin was measured by immunoprecipitation, SDS polyacrylamide gel electrophoresis and fluorography using 4 × 107 cpm of [35 S]methionine-labeled protein. Experiments were repeated three times, and the results were reproducible. (F) Optimal computer folding at the region of the initiation codon of p27Kip1 mRNA was performed by the method of Zuker (2003).

for 20 min; actin and ␥-tubulin, treated with 1 ml of 100% methanol at −20 ◦ C for 2 min; and polyamines (spermidine/spermine), incubated with 0.1 ml of PBS containing 1% glutaraldehyde and 0.1% Na2 S2 O5 at 37 ◦ C for 30 min followed by PBS containing 2 mg/ml NaBH4 at 37 ◦ C for 10 min. Cells were then incubated with 0.1 ml of PBS containing 3% bovine serum albumin, 0.2% Triton X-100 and antibodies shown below at 22 ◦ C for 60 min: anti-actin, SC-1616 (Santa Cruz); anti-vimentin, Clone V9 (Neo Markers); anti-␣-tubulin, SC-5286 (Santa Cruz); anti-␥-tubulin, CLONEGTU-88 (Sigma–Aldrich); and anti-spermidine/spermine, ASPM-29 (Fujiwara and Masuyama, 1995). After washing cells with PBS containing 0.1% Triton X-100, cells were further treated with anti-mouse IgG antibody (Alexa Fluor® 488, Molecular Probe) and then DAPI (4 ,6-diamino-2-phenylindole) in the dark. Cells were analyzed using a fluorescence microscope (Olympus® BX51/DP70) after treatment with ProLong® Antifade Kit (Molecular Probe). 2.8. Assay for polymerization of actin The assay was performed according to the method of OriolAudit (1978) with some modifications. The reaction mixture (0.4 ml), containing 20 mM Hepes-KOH (pH 7.5), 50 ␮g actin (non-muscle, Cytoskeleton Inc.), 0.5 mM ATP, 50 mM KCl, 0.5 mM 2-mercaptoethanol, 3 mM sodium azide, 5% sucrose, and MgCl2 and spermine shown in the figure, was incubated at 37 ◦ C for 30 min. After the incubation, the reaction mixture was centrifuged at 100 000 × g at 4 ◦ C for 3 h. The pellet was solubilized with 0.1 M NaOH,

and the concentration of the polymerized actin was measured by the method of Bradford (1976).

3. Results 3.1. Effect of polyamine deficiency on G1 to S phase transition To study the effects of polyamines on the G1 to S phase transition, HeLa cells were synchronized at G1 by treatment with 2 mM thymidine for 24 h. Cells were then cultured in the absence or presence of the inhibitors of polyamine biosynthesis (0.5 mM DFMO and 0.1 mM EGBG) (Fig. 1A), and progression of the cell cycle was analyzed. As shown in Fig. 1B, the delay of transition from G1 to S phase was not significant at 12 h (percentage of cells at G1 phase, 47.8% vs. 41.3%) after the addition of DFMO and EGBG although polyamine content started to decrease significantly (Fig. 1C). A delay of transition from G1 to S phase was clearly observed at 18 and 24 h, indicating that polyamines play important roles at the G1 /S boundary. The levels of protein kinases and their regulatory proteins involved in the G1 to S phase transition were examined by Western blot analysis. As shown in Fig. 2A, the levels of p27Kip1 and p21Cip1/WAF1 , were increased at 24 h after the addition of DFMO and EGBG. The increase in both proteins was parallel with the delay of progression of the cell cycle. Levels of eight other proteins [cyclin D1, cyclin E, CDK2, CDK4, Rb, p16INK4a , PCNA (Pines, 1994) and

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CDC34 (Pintard and Peter, 2003)] were not altered by polyamine depletion. Since the effects of polyamine depletion on the level of p27Kip1 were more pronounced than on the level of p21Cip1/WAF1 , the mechanism of increase in p27Kip1 was studied at 24 h after addition of DFMO and EGBG. The level of p27Kip1 mRNA was higher in control cells compared to polyamine-deficient cells (Fig. 3A), suggesting that the increase in level of p27Kip1 in polyamine-deficient cells is post-transcriptional. It has been reported that stability of p27Kip1 protein is regulated by phosphorylation of p27Kip1 which becomes a signal for ubiquitination (Ishida et al., 2000). Thus, the level of phosphorylation of p27Kip1 in normal and polyamine-deficient cells was compared by 2D gel electrophoresis followed by immunoblotting. There are two phosphorylation sites (T157 and T198) in p27Kip1 , and the unphosphorylated form of p27Kip1 was the predominant species (Fig. 3B) (Ciarallo et al., 2002). The phosphorylation state of p27Kip1 was very similar in control and polyamine-deficient cells (Fig. 3B), suggesting that stability of p27Kip1 is not influenced by polyamines. The stability of p27Kip1 was studied in the presence of cycloheximide (10 ␮g/ml). Although the percentage of the remaining p27Kip1 at 1 h after incubation with cycloheximide was higher in polyamine-deficient cells than in control cells, p27Kip1 was stable during incubation from 1 h to 4 h under both conditions (Fig. 3C). Furthermore, the level of E3 proteins [Kip1 and 2 ubiquitination promoting complex (KPC1 and 2) (Kamura et al., 2004), p45SKip2 (Sutterluty et al., 1999), and p53 inducible protein with Ring H2 domain (Pirh2) (Hattori et al., 2007)] involved in the degradation of p27Kip1 was nearly equal in both polyamine deficient and normal cells (Fig. 3D), although a modest decrease in p45Skp2 was observed in polyamine-deficient cells. The effect of polyamine deficiency on the synthesis of p27Kip1 was then examined by labeling of p27Kip1 with [35 S]methionine. As shown in Fig. 3E, the synthesis of p27Kip1 was enhanced by polyamine deficiency, whereas the synthesis of actin was slightly decreased by polyamine deficiency. It has been reported that polyamines inhibit the scanning of 40S ribosomal subunits on the mRNA consisting of the double-stranded RNA through polyamine stabilization of its RNA structure (Shimogori et al., 1996). This was the case in the 5 -UTR of p27Kip1 mRNA, which has extremely long 5 -UTR consisting of 600 nucleotides (Polyak et al., 1994) and forms the very stable double-stranded RNA at the region of the initiation codon (Fig. 3F). These results support an idea that synthesis of p27Kip1 is stimulated in the absence of polyamines at the level of translation.

3.2. Effect of polyamine deficiency at G2 /M phase Cells were first synchronized at the G1 phase by treatment with 2 mM thymidine for 24 h, and then cultured in the absence or presence of DFMO and EGBG for 12 h, followed by the addition of aphidicolin (5 ␮g/ml) to inhibit DNA synthesis (see Fig. 1). Thus, the effect of polyamine deficiency on the transition from the G2 /M phase to the G1 phase could be evaluated. As shown in Fig. 1B, a delay of the transition from G2 /M to G1 was observed by in response to polyamine deficiency at 24 h after the addition of DFMO and EGBG. The percentage of cells at the G2 /M phase was about 10% higher in polyamine-deficient cells than that in normal cells. The results indicate that polyamines also play important roles at G2 /M phase. However, the involvement of polyamines at the G2 /M phase was less prominent than that at the G1 /S boundary. The levels of protein kinases and their regulatory proteins involved in the G2 /M to G1 phase transition were examined by Western blot analysis. The levels of eight kinds of proteins [cyclin A, cyclin B, CDK1, CDK2 (Pines, 1994), Chk1 (Walworth, 2001), CDC25B (Nilsson and Hoffmann, 2000), Mad2 (Chen et al., 1996),

Fig. 4. Distribution of spermine and spermidine in HeLa cells at interphase and at various stages of M phase. HeLa cells were cultured as shown in Fig. 1A. At 12 h after the addition of aphidicolin, HeLa cells were stained with ASPM-29 mAb and DAPI. DIC, differential interference contrast. Cells at interphase and those at metaphase, anaphase and telophase of M phase are shown. SPM, spermine; SPD, spermidine. Bar, 20 ␮m.

and CDC27 (Georgi et al., 2002)] were very similar in control and polyamine deficient cells (see Fig. 2). We next examined polyamine distribution at the M phase to estimate the function of polyamines at M phase by immunocytochemistry using an anti-spermidine/spermine antibody (Tanabe et al., 2004). It is known that polyamines usually exist in the cytoplasm through their binding to RNA, mainly associated with ribosomes (Tanabe et al., 2004; Watanabe et al., 1991). Since DNA condensation and cytokinesis occur dynamically at M phase, the possibility that polyamines may shift from cytoplasm to nuclear DNA after disappearance of the nuclear membrane was examined. As shown in Fig. 4, spermidine and spermine were mainly located in cytoplasm through metaphase, anaphase and telophase. The results suggest that polyamines are involved in cytokinesis rather than DNA condensation at the M phase. To study the effects of polyamines on cytokinesis, the structure of actin was first examined, because it has been reported that polymerization of actin is enhanced by polyamines (Oriol-Audit, 1978; Pohjanpelto et al., 1981). Polymerization of actin was enhanced by 0.5–1 mM spermine in the presence of 0.5–1 mM Mg2+ and absence of Ca2+ (Fig. 5A). As shown in Fig. 5B, actin forms the contractile ring at telophase (Yuce et al., 2005) in the presence of polyamines. When HeLa cells were treated with DFMO and EGBG for 36 h, an unusual structure of actin fibers was observed (Fig. 5C), and the number of multinucleate cells increased by 3-fold (Fig. 5C and D). Vimentin is one of the components of intermediate filaments and is associated with actin filaments. Distribution of vimentin at the M phase was similar to that of actin, and an unusual structure of vimentin fibers was observed in response to polyamine-deficiency (data not shown). The results indicate that cytokinesis is more strongly influenced by polyamines at the M phase rather than chromatin structure (Snyder, 1989). Microtubules are a component of the spindle fiber (Yuce et al., 2005), so microtubule dynamics were followed by staining ␣tubulin. As shown in Fig. 6A, spindle fiber formation was clearly

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Fig. 5. Increase in multinucleate cells by polyamine deficiency and its relationship to the structural change of actin at G2 /M phase. (A) Effect of spermine on polymerization of actin in a cell-free system. Actin polymerization was determined as described in Section 2. Various concentrations of Mg2+ and spermine shown in the figure were added to the reaction mixture. Percentage of polymerized actin is shown as means ± S.E. of triplicate determinations. (B) Actin and DNA in normal cell cycle. HeLa cells were cultured as shown in Fig. 1A. At 12 h after the addition of aphidicolin, immunocytochemical analysis was performed using anti-actin and DAPI. Bar, 20 ␮m. (C) Structure of actin in the cells treated with DFMO and EGBG. HeLa cells were cultured as described above, except that DFMO and EGBG were added as shown in Fig. 1A. Bar, 20 ␮m. (D) Increase in the number of multinucleate cells cultured with DFMO and EGBG. The number of multinucleate cells among 100 cells is expressed as the mean ± S.E. of triplicate determinations.

observed at telophase in the presence of polyamines. However, an unusual pattern of ␣-tubulin was observed in multinucleate cells generated by polyamine deficiency (Fig. 6A). The altered microtubules are probably caused by inhibition of polymerization of ␣-tubulin (Banan et al., 1998; Savarin et al., 2010). The centrosome is located at the microtubule-organizing center, and normally consists of a pair of centrioles. Centrioles were analyzed by staining ␥-tubulin which is one of the components of the pericentriolar matrix (Guan et al., 2008). As shown in Fig. 6B, a

pair of centrioles was observed at the periphery of the chromosome in single- and multi-nucleate normal cells. However, in polyamine deficient cells, unpaired centrioles and more than one pair of centrioles were occasionally observed. The percentage of cells having unusual centrioles was more than 2-fold higher in polyamine deficient cells than in control cells (data not shown). These results, taken together, indicate that progression of cytokinesis is delayed by polyamine deficiency through the structural change of actin, vimentin and ␣-tubulin.

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Fig. 6. Change of structure of ␣-tubulin and distribution of ␥-tubulin in cells by polyamine deficiency at G2 /M phase. (A) ␣-Tubulin and DNA in normal cells and cells treated with DFMO and EGBG. (B) Cellular distribution of ␥-tubulin in single nucleate cells and multinucleate cells. HeLa cells were cultured as shown in Fig. 1A. At 12 h after the addition of aphidicolin, immunocytochemical analysis was performed using anti-␣-tubulin, anti-␥-tubulin and DAPI. Bar, 20 ␮m.

4. Discussion To study the role of polyamines in progression of the cell cycle, we used synchronized cells, and DFMO and EGBG as inhibitors of polyamine biosynthesis. Under these conditions, the levels of all three polyamines were reduced. The effects of polyamines on cell growth are mainly dependent on the stimulation of specific kinds of protein synthesis (Igarashi and Kashiwagi, 2010). The optimal concentrations of putrescine, spermidine and spermine necessary for stimulation of protein synthesis in a cell-free system were 8–10 mM, 0.4–0.6 mM and 0.08–0.1 mM, respectively (Ogasawara et al., 1989). Thus, it is important to decrease the level of spermine to clarify the function of polyamines. At the G1 to S phase transition, the synthesis of several kinds of proteins such as Cct2 (T-complex protein 1, ␤-subunit), which are important for cell growth, is enhanced (Igarashi and Morris, 1984; Nishimura et al., 2009). Furthermore, polyamines decreased the level of inhibitors of cyclin dependent protein kinases CDK2 and CDK4: p21Cip1/WAF1 in MALME-3M cells (Kramer et al., 2001), p27Kip1 in CEM leukemia cells (Choi et al., 2000), and p21Cip1/WAF1 , p27Kip1 and p53 in IEC-6 cells (Ray et al., 1999). In this study, the mechanism of the increase in p27Kip1 in polyamine-deficient HeLa cells was analyzed at the molecular level. Polyamine deficiency enhanced the synthesis of p27Kip1 which was measured by labeling of p27Kip1 protein with [35 S]methionine. The size of the 5 -UTR of p27Kip1 mRNA is the extremely long consisting of the 600 nucleotides (Polyak et al., 1994), and the scanning of 40S ribosomal subunits from the m7 G-cap to the

initiation codon AUG was inhibited by polyamines (Shimogori et al., 1996). Thus, synthesis of p27Kip1 was enhanced in the absence of polyamines. This is the first report to clarify the molecular mechanism for enhanced level of p27Kip1 in the absence of polyamines. Effect of polyamine deficiency at G2 /M phase was also examined using synchronized cells. If unsynchronized cells were used, the effect of polyamine deficiency was mainly observed at G1 to S transition because major portions of cells exist at G1 phase as a consequence of polyamine deficiency. Our strategy to use synchronized cells made it possible to examine the effect of polyamine deficiency at the G2 /M phase. The results show that transition from G2 /M to G1 phase is delayed by polyamine deficiency. We confirmed that polymerization of actin is enhanced by polyamines (Oriol-Audit, 1978; Pohjanpelto et al., 1981), which is especially important at G2 /M phase (see Fig. 5). Polymerization of actin may be stimulated by its phosphorylation by spermine through the ternary complex formation of spermine-ATP-Mg2+ (Meksuriyen et al., 1998), because polyamines did not bind directly to actin (data not shown). Our results clearly indicate that both formation of contractile ring by actin and spindle fiber formation by ␣-tubulin were significantly inhibited by the decrease in polyamine content at the G2 /M phase in a cell culture system. It is also noted that synthesis of Cct2 is enhanced by polyamines (Nishimura et al., 2009), because Cct2 is a chaperonin located in cytoplasm and assists in the folding of actin, tubulin and several other proteins (Yokota et al., 1999). Accordingly, cytokinesis is delayed by polyamine deficiency through structural change of several kinds of proteins. The increase

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in ornithine decarboxylase at the G2 /M transition is probably necessary for cytokinesis at anaphase and telophase. Essentially, the same results were obtained using NIH3T3 cells and mouse mammary carcinoma FM3A cells (data not shown), suggesting that polyamine effect on progression of cell cycle is universal in all cell types. It has been also reported that DNA elongation rate is reduced in polyamine-deficient cells (Laitinen et al., 1998; Oredsson et al., 1990). Furthermore, the decrease in the activities of DNA polymerase ␣ (Igarashi and Morris, 1984; Koza and Herbst, 1992) and thymidine kinase (Igarashi and Morris, 1984) has been reported. Thus, polyamines play important roles in all the stages of the cell cycle.

Acknowledgements We thank Dr. K. Williams for his help in preparing the manuscript. We also thank Dr. K.-i. Nakayama, Aventis Pharma and Torii Pharmaceutical Co. for providing antibodies against KPC1 and KPC2, DFMO and FUT-175, respectively. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

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