The change of antizyme inhibitor expression and its possible role during mammalian cell cycle

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / y e x c r

Research Article

The change of antizyme inhibitor expression and its possible role during mammalian cell cycle Yasuko Murakami a,b , Jun-ichiro Suzuki a , Keijiro Samejima a , Kenjiro Kikuchi b,1, Tomasz Hascilowicz b,2 , Noriyuki Murai b , Senya Matsufuji b , Takami Oka a,⁎,3 a b

Research Institute of Pharmaceutical Sciences, Musashino University, 1-1-20 Shinmachi, Nishi-Tokyo, Tokyo 202-8585, Japan Department of Molecular Biology, The Jikei University School of Medicine, Minato-ku, Tokyo 105-8461, Japan

A R T I C L E I N F O R M AT I O N

AB ST R AC T

Article Chronology:

Antizyme inhibitor (AIn), a homolog of ODC, binds to antizyme and inactivates it. We report here

Received 4 March 2009

that AIn increased at the G1 phase of the cell cycle, preceding the peak of ODC activity in HTC cells

Revised version received

in culture. During interphase AIn was present mainly in the cytoplasm and turned over rapidly

24 April 2009

with the half-life of 10 to 20 min, while antizyme was localized in the nucleus. The level of AIn

Accepted 25 April 2009

increased again at the G2/M phase along with ODC, and the rate of turn-over of AIn in mitotic cells

Available online 6 May 2009

decreased with the half-life of approximately 40 min. AIn was colocalized with antizyme at centrosomes during the period from prophase through late anaphase and at the midzone/

Keywords:

midbody during telophase. Thereafter, AIn and antizyme were separated and present at different

Antizyme inhibitor

regions on the midbody at late telophase. AIn disappeared at late cytokinesis, whereas antizyme

Antizyme

remained at the cytokinesis remnant. Reduction of AIn by RNA interference caused the increase in

ODC

the number of binucleated cells in HTC cells in culture. These findings suggested that AIn

Cell cycle

contributed to a rapid increase in ODC at the G1 phase and also played a role in facilitating cells to

Centrosome

complete mitosis during the cell cycle.

Cytokinesis

Introduction Polyamines are indispensable for cell growth, but they display severe cytotoxic effects if they accumulate in excess [1–3]. The cellular concentrations of polyamines are rapidly and tightly regulated by the multiple factors involved in the biosynthesis and transport of polyamines. Mammalian ornithine decarboxylase (ODC) is the first and key enzyme in the polyamine biosynthetic pathway [4]. This enzyme has a very short half-life and is highly regulated [5–8]. ODC is induced by various growth stimuli and degraded rapidly when polyamine levels increase. Polyamineenhanced ODC degradation is mediated by antizyme, an inhibitory

© 2009 Elsevier Inc. All rights reserved.

protein of ODC induced by polyamines. Antizyme binds to ODC and targets it to the 26S proteasome for ubiquitin-independent degradation [9]. In addition, antizyme serves to lower polyamine contents by suppressing the uptake of extracellular polyamines [10,11] and accelerating the excretion of intracellular polyamines [12]. Maintaining the balance between ODC and antizyme appears to be important for normal cell growth since ODC overproduction is associated with neoplastic transformation [13–15]. Furthermore, overexpression of antizyme has been shown to inhibit cell growth or to increase apoptosis [16–19]. Mammalian cells possess another putative regulatory molecule, antizyme inhibitor (AIn) that can play a role in the antizyme-

⁎ Corresponding author. Fax: +81 3 3295 0140. E-mail addresses: [email protected], [email protected] (T. Oka). 1 Present address: Department of Pediatrics, The Jikei University School of Medicine, Minato-ku, Tokyo 105-8461, Japan. 2 Present address: Dai3 Hospital, The Jikei University School of Medicine, Komae-shi, Tokyo 201-8601, Japan. 3 Present address: Wakunaga Pharmaceutical Institute, Chiyoda-ku, Tokyo 101-0062, Japan. 0014-4827/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2009.04.024

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mediated feedback system for polyamine homeostasis. AIn is the product of an ODC-related gene but lacks enzymatic activity [20,21]. It binds to antizyme with a higher affinity than does ODC, thereby releasing active ODC from the ODC–antizyme complex [22,23]. AIn also inhibits ODC degradation by the 26S proteasome [20]. These properties of AIn suggest that AIn functions as a positive regulator of both ODC and polyamine transport by trapping antizyme in cells. Indeed, it has been shown that AIn overexpression results in elevation of ODC activity and polyamine uptake and increases growth rate [24]. Moreover, AIn overproducing cells give rise to tumors when injected into nude mice [24], while reduction of AIn levels by AIn siRNA reduces the intracellular concentration of polyamines and inhibits cell proliferation [25,26]. Recently, AIn and antizyme have been found to regulate centrosome duplication [27]. More recently, it was reported that AIn homozygous mutant mice die on the first postnatal day and exhibited reduced cellular levels of putrescine and spermidine and abnormal liver morphology [28]. These findings suggest that AIn is an important regulator of polyamine metabolism and cell growth. At present, however, the regulation and cellular function of AIn have not been fully elucidated partially due to its low intracellular content. AIn is even less abundant than ODC, a low abundant protein [23]. In rat liver, AIn is induced after feeding diet but its maximum amount is less than 10% of the ODC maximal and is roughly comparable to that of antizyme [29]. Like ODC, AIn is a short-lived protein [29] in vivo, but unlike ODC, AIn is degraded in a ubiquitin-dependent manner and stabilized by antizyme [30]. To understand how AIn works coordinately with ODC and antizyme, it deemed important to examine when and where AIn is expressed and interacts with antizyme during the cell cycle. We also investigated the regulatory properties and possible role of AIn during the cell cycle.

Materials and methods Cell culture, cell cycle synchronization and transfection HTC cells were maintained in Dulbecco's modified Eagle's minimum essential medium (DMEM, GIBCO) supplemented with 2% fetal bovine serum, 4% newborn calf serum. To obtain cells arrested at promethaphase, exponentially growing cells were treated with 400 ng/ml nocodazole for 16 h. Cells were collected by either pipeting or by gentle shaking, washed twice with PBS and further cultivated for the indicated times in complete DMEM without nocodazole. To synchronize cells to the G1/S boundary, double thymidine block was done by incubating HTC cells in the culture medium containing 2 mM thymidine for 16 h. Cells were washed with PBS and then incubated for 8 h in fresh complete DMEM without thymidine. Cells were incubated again for 13 h in 2 mM thymidine. After washing twice with PBS, cells were cultured in fresh medium to enter the cell cycle. Transfection of AIn siRNA (Ambion 199244 for HTC cells, Qiagen 68650 for HC11 cells) or control siRNA (Qiagen) was performed using lipofectamin 2000 as instructed by the manufacturer (Invitrogen).

Flow cytometric analysis Cells were trypsinized, washed with PBS, and centrifuged for 5 min at 1000 g. Washed cells were incubated with 20 μg/ml, propidium iodide (Sigma) dissolved in PBS with 0.2 mg/ml RNase A (Sigma)

and 0.1% (v/v) Triton X-100 for 30 min at 37 °C. DNA content was measured by flow cytometry (FACSAria, Becton Dickinson).

Immunofluorescence microscopy The following antibodies were used: HI-12 monoclonal antibody to rat AIn (40 μg/ml) [29], polyclonal anti γ-tubulin antibody (40 μg/ml, Sigma), polyclonal anti-antizyme antibody (40 μg/ml) [31]. Secondary antibodies were Alexa Fluor 488 or 568 conjugated anti-mouse IgG or anti-rabbit IgG (1:300, Molecular Probes). Cells were fixed in 4% (v/v) paraformaldehyde-PBS for 30 min at room temperature. After being washed with PBS, cells were treated with 3% BSA in PBS containing 0.1% Triton X-100 for 20 min. Cells were then incubated overnight at 4 °C with the primary antibody, washed four times with PBS containing 0.1% Tween 20 and then incubated for 20 min at room temperature with secondary antibody (1:300), TO-PRO 3 (1:200, Molecular Probe) was used to stain nuclei and rhodamine–phalloidine (1:50, Cytoskelton) was used to stain F-actin. The antibodies, TO-PRO-3 and rhodamine– phalloidine were diluted in the same solution as used for blocking. The images were acquired with the Olympus FV1000 Laser Microscope using ×60 oil-immersion objective lens (UPlan FL N 60×/1.30).

Western blot analysis After three freeze–thaw cycles, cells were suspended in 0.3 ml of 25 mM Tris/HCl (pH7.5) containing 2 mM DTT, 0.01% Tween 80 and proteinase inhibitor cocktail (Roche). The cell suspension was sonicated for 20 s and centrifuged at 12,000 g for 20 min at 4 °C. Protein concentration was determined using BCA protein assay reagent (Pierce) and 20 μg–60 μg of the supernatant protein was separated on 11.5% polyacrylamide-SDS gel. Immunoblotting was performed according to a standard protocol using Hybond-P membrane (GE Healthcare). Immunodetection was carried out with the primary antibody i.e. HI-12 monoclonal antibody to rat AIn at 260 ng/ml, monoclonal antibody to cyclin B1 (MBL, at 100 ng/ml) and polyclonal anti-β-actin antibody (Cell Signaling, 1:1000) and the horseradish peroxidase-conjugated secondary antibody (Histofine, Nichirei) at a dilution of 1:200,000. The proteins were visualized using ECL-Advance (GE Healthcare), and the bands were quantified by using the Fuji LAS-3000 system (Fuji Photo Film) with Multi Gauge software. Non-specific band or Direct Blue (ALDRICH) stain was used as a loading control.

Determination of ODC and antizyme activities Cells were washed three times with PBS and disrupted by three freeze–thaw cycles. Then, extraction buffer (25 mM Tris/HCl, pH 7.4, 1 mM DTT and 0.01% Tween 80) was added, and the cell suspension was sonicated for 20 s and centrifuged at 18,000 g for 20 min at 4 °C. The supernatant was saved for assay of ODC and antizyme activities. The supernatant contained free ODC as well as ODC bound to antizyme in 1:1 molecular ratio. The free ODC activity in the supernatant fraction was assayed by measuring the release of 14CO2 from L-[1-14 C] ornithine (Moravak Biochemicals). The enzyme reaction mixture contained 0.0625 μCi of L-[1-14C] ornithine, 0.4 mM L-ornithine, 40 μM pyridoxal phosphate, 5 mM DTT, 40 mM Tris/HCl buffer, pH 7.4, 0.01% Tween 80 and enzyme sample solution in a final

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volume of 125 μl. One unit of ODC activity was defined as the amount forming 1 nmol of CO2/hour at 37 °C and expressed as units/mg protein. The ODC activity bound to antizyme could be released by AIn [20]. This was done by mixing recombinant AIn (0.1 μg) [20] with the enzyme sample (supernatant) in the ODC reaction mixture prior to enzyme assay. The ODC activity released was then determined together with pre-existing free ODC activity as described above, which gave total ODC activity. The activity of antizyme was calculated by subtracting free ODC activity from total ODC activity since antizyme binds to ODC in 1:1 molecular ratio [32]. One unit of antizyme activity was defined as one unit of ODC activity.

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Results The antizyme inhibitor level fluctuates during the cell cycle To investigate the dynamics of cellular regulation of AIn, we analyzed the level of AIn during the cell cycle and compared it with that of ODC and antizyme. HTC cells were synchronized at the prometaphase by nocodazole treatment and then released into fresh medium. The cell cycle profile of released cells was analyzed by flowcytometry (Fig. 1 top) and the levels of AIn, antizyme and ODC were examined. As shown in Fig. 1A, most of cells (98%) were in the G2/M phase of the cell cycle at the time of

Determination of the synthetic rate of antizyme inhibitor in cells The rate of synthesis of AIn was determined by measuring the incorporation of 35S-radiolabelled methionine and cysteine into the protein. To induce AIn synthesis, HTC cells were first cultured in fresh DMEM for 2 h, and then the culture medium was replaced by RPMI 1640 without methionine and cysteine supplemented with 2% fetal bovine serum and 4% newborn calf serum. After incubation for 30 min the mixture of L-[35S] methionine and L-[35S]cysteine (12.5 μCi/ml, PerkinElmer) was added to the medium, and cells were incubated for 10 min before harvesting. Cells were washed with PBS containing 5 mM unlabeled methionine. After three freeze–thaw cycles, cells were suspended in 0.5 ml of buffer A (10 mM Tris/HCl buffer, pH 7.5, containing 0.1 mM EDTA, 0.5 mM DTT, 0.02% BSA, 0.1% Triton X-100, 0.1% SDS and 0.1% Tween 80) containing 5 mM methionine. The cell suspension was sonicated for 20 s and centrifuged at 16,000 g for 20 min at 4 °C. Using an aliquot of the supernatant, radioactivity in total protein was determined by precipitating with TCA. In order to determine the amount of radiolabelled AIn, aliquots containing equal amounts of total protein were incubated with HI-12 monoclonal anti-AIn antibody for 3 h at 4 °C. The antibody–AIn complex was precipitated by incubation with adsorbent (GE Healthcare) overnight at 4 °C with mixing by a rotary shaker. The precipitate was collected by centrifugation and washed four times with buffer A. The precipitates were suspended in Laemmli SDS-PAGE sample buffer, heated for 5 min at 100 °C and centrifuged at 15,000 g for 5 min. The supernatant was electrophoresed on 11.5% SDS-polyacrylamide gel, and the labeled protein was detected and quantitated using the Fla-2000 image analyzer and expressed as PSL (Photo Stimulated Luminescence).

Synthesis of antizyme inhibitor in reticulocyte lysate Total RNA was extracted from HTC cells, and poly(A+) RNA was isolated from the total RNA by using mRNA isolation kit (MicroFastTrack, Invitrogen). The poly (A+) RNA (1.0 μg) was heated at 65 °C for 10 min, and then translated in a total volume of 30 μl of the rabbit reticulocyte lysate translation system containing L-[35S] methionine and the indicated concentrations of putrescine or spermidine. Total protein synthesis was measured by essentially following the method of Kameji and Pegg [33]. AIn was immunoprecipitated from the translation assay mixture by the addition of HI-12 monoclonal anti-AIn antibody and protein A, separated on SDS-PAGE, and quantified as described above.

Fig. 1 – Temporal changes of AIn protein, ODC and antizyme activities in HTC cells after release from nocodazole block. HTC cells were synchronized at the G2/M phase by treatment with nocodazole for 16 h. Cells were then washed twice with PBS and replated in fresh complete DMEM (time: 0 h). Cells were harvested at the indicated times. Cell cycle progression was monitored by flow cytometry analysis and the percentage of cells in each phase of the cell cycle was shown on the top. Cell extracts were prepared and subjected to SDS-PAGE and Western blot analysis was performed with the anti-AIn antibody. The amount of AIn protein was determined using a lumino image analyzer (A). ODC and antizyme activities in the cell extract were determined as described in the Materials and methods and plotted against time after release from nocodazol block in (B) and (C), respectively. The mean maximum level of AIn at 2 h, ODC and antizyme activity at 4 h was set to 100%. Each column with the corresponding standard deviation represents the data from 3–6 independent experiments.

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release and expressed a substantial level of AIn. The AIn level increased further shortly after being cultured in fresh medium, reaching a peak at 2 h in the early G1 period. Thereafter, the AIn level decreased steadily to approximately 20% of the peak level and remained low during the next 20 h (data not shown). The level of AIn mRNA changed in parallel with that of AIn protein, but the magnitude of increase was smaller, i.e. approximately 25% (data not shown). The ODC activity began to increase at 2 h reaching a peak at 4 h, and then decreased rapidly (Fig. 1B). Antizyme activity increased steadily and reached the maximum at 4 h, followed by the gradual decrease (Fig. 1C). The intracellular concentration of polyamines including putrescine, changed in the manner similar to the level of antizyme, reaching a small peak at around 4 h (data not shown). However, the change in AIn protein and ODC activity during the second G2/M could not be accurately determined because of the loss of synchrony at late times. To examine further the changes of AIn, ODC and antizyme during the period from S to G2/M phase, HTC cells were synchronized at the G1/S boundary by double thymidine treatment and then released into fresh medium. The cell cycle profile of released cells was analyzed by flow cytometry and the levels of AIn, cyclin B1, ODC and antizyme were examined. As shown in Fig. 2A, cells entered the G2/M phase in 4 h after the release. The level of AIn in cells arrested at the G1/S boundary was low but increased rapidly reaching a peak at 4 h (Fig. 2B). Then, the AIn level gradually decreased and started to increase again at 9–10 h. The ODC activity changed largely in parallel with AIn. The level of antizyme was very low, and thus it was difficult to co-relate its changes with that of AIn or ODC. Based on the Western blot analysis of cyclin B, a marker for the G2/M phase of the cell cycle, the first peak of AIn corresponded to the G2/M period (Fig. 2B).

Stability of AIn in the cells at interphase and mitosis To measure the half-life of AIn during the cell cycle, HTC cells were synchronized at the prometaphase by nocodazole treatment followed by cultivation in fresh medium. Immediately (mitosis) and 2, 4 or 6 h (interphase) after the incubation, the half-life of AIn was determined in the presence of cycloheximide (CHX) by examining the level of AIn by immunoblot (Fig. 3A) and quantification by a lumino image analyzer (Fig. 3B). During 4–6 h AIn decayed in two distinct phases: in the first phase AIn was degraded with the half-life of approximately 15 min while in the second phase AIn remained largely stable (Fig. 3B). On the other hand, AIn in mitotic cells (0 h) decayed slowly with the half-life of approximately 40 min (Fig. 3B). These results indicated the difference in the rate of AIn degradation between interphase and mitosis during the cell cycle. The results also indicated that there was no apparent co-relation between the levels of AIn and antizyme. In addition, we examined the degradation of ODC and found that the half-life of ODC degradation during 30 min of CHX treatment was 19 min, 20 min, 14 min and 7 min at 0, 2, 4, and 6 h, respectively (Fig. 3C).

Fig. 2 – Temporal changes of AIn protein, ODC and antizyme activities during the cell cycle. HTC cells were synchronized at the G1/S boundary by double thymidine block. Cells were then released from the block by being placed into fresh medium. Cells were harvested at the indicated times after the release from thymidine block. (A) Cells were collected, fixed, and stained with propidium iodide, and analyzed by flow cytometry. The percentage of cells in each phase of the cell cycle was plotted against time. (B) Cell extracts were prepared and subjected to SDS-PAGE and Western blot analysis with the anti-AIn antibody and anti-cyclin B1 antibody. A nonspecific cross-reacting band (NS) was used as a loading control. The level of AIn protein was determined using a lumino image analyzer and plotted against time. ODC and antizyme activities in the cell extract was determined as described in the Materials and methods and plotted. Results similar to those in (A) and (B) were obtained in two other experiments.

Subcellular localization of AIn changes during the cell cycle As shown in Fig. 4, AIn was present mainly in the cytoplasm during interphase, although it was sometimes detected at centrosomes as reported previously [27]. During the period from prophase through late anaphase, AIn was largely found in

centrosomes and then became concentrated on the midzone/ midbody during telophase and cytokinesis. At late cytokinesis AIn disappeared from the midzone/midbody (Fig. 4). During the

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period from prophase through cytokinesis, AIn always colocalized with γ-tubulin (Fig. S1), which is an established centrosomal marker and also known to associate with the mitotic spindle and midbody [34]. During interphase antizyme was present mainly in the nucleus but was not found at centrosomes (Fig. 4). Antizyme colocalized with AIn mainly at centrosomes during the period from prophase through late anaphase. During telophase and cytokinesis antizyme was concentrated on the midzone/midbody (Fig. 4). Both antizyme and AIn were present together at the midzone/midbody at telophase. Thereafter, however, antizyme restricted in the minus regions of the microtubule bundles, whereas AIn was largely move to the plus regions at late telophase and cytokinesis (Fig. 4). At the late phase of cytokinesis AIn fully dissociated from antizyme and disappeared. The above results indicated that both AIn and antizyme changed their localization during the cell cycle.

Effect of AIn reduction on cytokinesis To investigate the possible role of AIn in cytokinesis, we reduced the expression of endogenous AIn in HTC cells by AIn siRNA. Cells were harvested 48 h after the treatment with siRNA and subjected to immunofluorescence analysis. As shown in Fig. 5, AIn siRNA treatment suppressed the expression of AIn protein and increased the number of binucleated cells. Quantitative analysis revealed that the proportion of the binucleated cells in AIn-depleted culture increased to 5.0 ± 1.5% from 0.9 ± 0.9% (n = 3) in control culture (Fig. 5). However, during 48 h of siRNA treatment there were no apparent sign of other abnormalities. Similar results were obtained with HC11 cells, a normal mouse mammary cell line (Fig. S2).

Effect of polyamines on expression of AIn

Fig. 3 – The half-life of AIn protein in G1 and mitotic HTC cells. HTC cells were synchronized at the G2/M phase by treatment with nocodazole for 16 h. Mitotic cells were collected by gentle shaking, washed twice with PBS and replated in fresh complete DMEM without nocodazole. Immediately (0 h) or 2, 4 and 6 h after culture, cycloheximide (CHX) was added at the final concentration of 50 μg/ml and cells were harvested at the indicated times. (A) Cell extracts were prepared and subjected to SDS-PAGE and Western blot analysis with the anti-AIn antibody and anti-β-actin antibody. β-Actin or a nonspecific band from the membrane treated with Direct Blue 71 was used as a loading control. (B) The amount of AIn was quantified using a lumino image analyzer and presented as % of the initial value (at time 0 min). Each point with the corresponding standard error represents the data from two (2 h, 6 h) or three (0 h, 4 h) independent experiments. (C) Cell extracts were prepared and determined for total ODC activity. Details are in Materials and methods.

AIn, a homolog of ODC [20,21] and ODC activity changed in parallel during the cell cycle (Figs. 1, 2). ODC activity is negatively controlled by polyamines such as putrescine, spermidine and spermine [8], whereas α-difluoromethylornithine (DFMO), a potent enzymeactivated irreversible inhibitor of ODC, increases the amount of ODC protein [35]. Thus, it was of interest to examine their effects on the level of AIn. HTC cells were first cultured for 2 days and incubated for 6 h in fresh medium with or without DFMO (5 mM). Exposure of HTC cells to fresh medium rapidly increased AIn protein, and the presence of DFMO augmented the increase in AIn protein at 6 h (Fig. 6A). Addition of fresh medium also increased the intracellular level of putrescine, but DFMO greatly reduced its level (Fig. 6A). In contrast, addition of putrescine (10 mM) inhibited the increase in AIn protein at 2 and 6 h (Fig. 6B). We confirmed immunocytochemically the decrease of AIn expression in HTC cells treated with putrescine (data not shown). Putrescine also decreased the level of AIn when added at later times (data not shown). In addition to putrescine, spermidine, spermine and agmatine also inhibited the induction of AIn in a concentration-dependent manner (Fig. 6C). These results suggested that expression of AIn was negatively regulated by the polyamines. We next examined whether the down-regulation of AIn by putrescine was due to the degradation of AIn protein by measuring the half-life of AIn protein. HTC cells were cultured for 2 h in fresh medium with or without putrescine and then cycloheximide (CHX) was added. As shown in Fig. 6D, the decay of

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Fig. 5 – Effect of AIn reduction on cytokinesis. HTC cells were transfected with control siRNA or AIn siRNA as described in the Materials and methods. After 48 h, cells were fixed and stained for AIn (green), F-actin (red) and nuclei (blue) and analyzed for binucleated cells. Bar, 10 μm. The results are from three independent cell culture experiments. In each case at least 100 cells were examined and the percent of binucleated cells in culture is presented. Error bars, SD.

AIn in the first 30 min occurred with the similar rate with or without putrescine, although putrescine slightly decreased the decay rate during the second phase (Fig. 6D). In addition, putrescine did not inhibit the increase in AIn mRNA that occurred within 2 h after exposure to fresh medium (data not shown). The effects of putrescine on the incorporation of labeled amino acids into AIn polypeptide in HTC cells and on the translation of AIn mRNA in a cell-free system were also examined. Putrescine significantly inhibited both the synthesis of AIn in HTC cells (Table 1) and the translation of AIn mRNA in cell-free system (Fig. 7A). On the other hand, the inhibitory effect of putrescine on total protein synthesis was much less in both cases. Spermidine was more effective in inhibiting the in vitro translation of AIn, although its effect on total protein synthesis was also large (Fig. 7B). These results are consistent with the view that polyamines can negatively control AIn synthesis acting at the level of translation.

Discussion We showed here that AIn protein increased in both early G1 and G2/M phases of the cell cycle together with ODC activity (Figs. 1 and 2). The study of AIn stability indicated that the initial decay rate of AIn was faster in cells during interphase

than in mitotic cells (Fig. 3). In addition, immunofluorescence studies showed that during interphase AIn was present mainly in the cytoplasm, while antizyme was localized in the nucleus (Fig. 4). In contrast, during the period from prophase through late anaphase AIn became colocalized with antizyme at centrosomes. These results indicated that AIn changed its level along with the altered rate of decay and subcellular location during the cell cycle. Earlier studies showed that ODC activity increased in a biphasic manner during cell cycle [7] and that AIn increased preceding ODC in mouse fibroblasts and in rat liver following the growth stimuli [21,29]. In this study we obtained the similar results in HTC cells during the cell cycle. The faster induction of AIn may inhibit antizyme, thereby extending the half-life of ODC and allowing a rapid increase in ODC. This view is supported by the observation that growth stimuli caused both the decrease in ODC–antizyme complex and the increase in ODC activity in cultured cells as well as in rat liver [29,36]. Furthermore, using HTC cells expressing glucocorticoid-inducible AIn, we found that induction of AIn by dexamethasone led to the decrease in the antizyme activity and the increase in the half-life of ODC and ODC activity (unpublished results). These results are consistent with the above view that AIn allows induction of ODC more readily. ODC is the first and rate-limiting enzyme in the biosynthesis of polyamines. ODC turns over rapidly. Antizyme, a key regulator

Fig. 4 – Changes in the cellular localization of AIn during the cell cycle. Cells were synchronized at the G1/S boundary by double thymidine block. Cells were then released from the block by being placed into fresh medium and harvested at the indicated phases of cell cycle after the release from thymidine block. Nuclei were stained with TO-PRO-3 (blue). AIn (red) and antizyme (green) were stained with each antibody, respectively. Scale bars: 10μm.

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Fig. 6 – Effects of DFMO and polyamines on the induction and the decay rate of AIn. (A) HTC cells were cultured in fresh medium with or without DFMO (5 mM) and harvested at 0 and 6 h. The level of AIn protein was analyzed by SDS-PAGE/Western blotting. A nonspecific band from the membrane treated with Direct Blue 71 was used as a loading control. Putrescine contents were determined [49] at the indicated times. ⁎ND: Not detectable (B) HTC cells were grown to approximately 60% confluency, and the culture medium was replaced with fresh medium with or without putrescine (10 mM). Cells were cultured for the indicated times. Cell extracts were prepared and subjected to SDS-PAGE and Western blot analysis with the anti-AIn antibody. A nonspecific band from the membrane treated with Direct Blue 71 was used as a loading control. The amount of AIn was quantified using a lumino image analyzer and shown as % of the initial value (at time 0 h) without putrescine. (C) HTC cells were cultured in fresh medium with or without the indicated polyamine or agmatine at the concentrations indicated. After 6 h cells were harvested and the level of AIn protein was analyzed by SDS-PAGE/Western blotting. The amount of AIn protein was determined using a lumino image analyzer and shown as % of control. (D) AIn was induced by feeding with fresh medium for 2 h with (■) or without (□) putrescine (10 mM) and then cycloheximide (50 μg/ml) was added. Cells were incubated for 0, 10, 20, 30 and 50 min. The level of AIn remaining at indicated times was determined by SDS-PAGE and Western blotting. The intensity of AIn band was determined using a lumino image analyzer and shown as % of the initial value (at time 0 min). The results are from three independent cell culture experiments. Error bars indicate SD.

of ODC, binds to ODC and targets it to ubiquitin-independent degradation by the 26S proteasome. Previous studies showed that antizyme moves between the nucleus and the cytoplasm, suggesting that it is involved in shuttling of ODC [37–39]. Antizyme itself is negatively regulated by AIn [22] and both AIn and antizyme are degraded in a ubiquitin-dependent manner [30,40]. Interestingly, antizyme and AIn were found to stabilize each other in cells co-transfected with antizyme and AIn cDNAs [30,40]. This mutual stabilization requires their binding [30,40]. In this study we found that AIn and antizyme were colocalized from prophase to late telophase, but they were separately present during interphase (Fig. 4) and that the half-life of AIn increased in mitotic cells as compared with that in cells during interphase. Thus it is possible that AIn and antizyme interact to form the stable complex during progression of mitosis, while AIn was less stable when it was separated from antizyme during interphase.

The dynamic change in the subcellular localization of both AIn and antizyme during the cell cycle suggested that their functions may vary depending on the different phases. As mentioned above, one of the possible roles of AIn during interphase may be to stabilize ODC and to increase its activity. This would make it possible to increase the cellular polyamine level promptly by growth stimuli that in turn may serve as a signal to trigger events leading to cell proliferation. In addition, it is to be noted that during interphase both AIn and antizyme are concentrated at centrosome and that alterations in the antizyme/AIn ratio cause the defect in centrosome numbers [27]. The authors hypothesized that antizyme regulates the stability of some centrosomal component(s) such as Aurora-A that is needed for the amplification of centrosome. It was shown more recently that antizyme promotes the degradation of Aurara-A mediated by Aurora-A kinase interacting protein 1 through the ubiquitin-independent pathway and that AIn inhibits this degradation [41]. In this study, however, we could not

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Table 1 – Effect of putrescine on incorporation of [35S] methionine and [35S] cysteine into AIn protein. 35

Culture

S incorporation

Addition AIn protein (PSL/dish) Total protein (×105 cpm/dish) None Putrescine

2575 ± 200 1838 ± 300⁎

138 ± 8 140 ± 13

HTC cells were cultured in fresh medium with or without 10 mM putrescine for 2.5 h and then allowed to incorporate [35S] methionine and [35S]cysteine for 10 min. The radioactivities incorporated into AIn and total soluble proteins were determined as described in the Materials and methods. Results are means ± S.D for three culture dishes. The asterisk (⁎) denotes significant difference (p < 0.05) between putrescine-treated and untreated cells.

detect antizyme in centrosome during interphase (Fig. 4). This may be due to the difference in culture conditions or anti-antizyme antibody used. AIn was found to colocalize with antizyme on the midzone/ midbody during telophase, but thereafter it was dissociated from antizyme in late telophase (Fig. 4) and disappeared at late cytokinesis. On the other hand, antizyme persisted at the cytokinesis remnant (Fig. 4). Dissociation of AIn from antizyme may result in the ubiquitination and degradation of AIn during cytokinesis. Recently Mukai et al. [42] reported the presence of deubiquitylating enzymes at the central spindle during cytokinesis. Further studies are needed to elucidate the dissociation mechanism of AIn from antizyme at late mitosis and to assess the possible role of these enzymes in AIn degradation. The importance of AIn in mitosis and cytokinesis was supported by the findings that reduction of AIn by RNA interference caused the increase in the number of binucleated cells in both HTC cells (Fig. 5) and HC11 cells (Fig. S2). This raises the question as to how AIn exerts its effect on the mitotic progression. Since the main function of AIn is presumably to bind and inhibit antizyme, its effect is likely to be mediated through its interaction with antizyme. Antizyme is known to be a multifunctional protein [6,32] and facilitates proteolysis of some proteins in a ubiquitinindependent manner. Possible candidate proteins being degraded via this pathway may include ODC and Aurora-A. These proteins may be colocalized with antizyme and AIn until the near end of mitosis before they get degraded. Since ODC could not be detected in centrosomes and midzone/midbody during mitosis (data not shown), it can be excluded as a possible candidate. On the other hand, it is noteworthy that the localization pattern of Aurora-A in HeLa cells [43,44] is similar to that of AIn during mitosis (Fig. 4). If we assume that Aurora-A is a target molecule of antizyme during mitosis, it is possible to speculate that dysfunction of the centrosome is responsible for the failure of cytokinesis in AIn-depleted cells, since centrosomes are required for completion of cytokinesis [45,46] and since Aurora-A has been implicated in the progression of mitotic events [47]. Further studies are clearly needed to evaluate the above idea and elucidate target molecules that are degraded via an antizyme-dependent mechanism during mitosis. We found that polyamines inhibited the induction of AIn and also lowered the pre-existing level of AIn, whereas an inhibitor of ODC, DFMO increased the level of AIn protein (Fig. 6). We also found that putrescine inhibited the synthesis of AIn polypeptide (Table 1) but did not alter either the level of AIn mRNA or the

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decay rate of AIn in HTC cells. Moreover we showed that putrescine significantly inhibited translation of AIn in a cell-free system (Fig. 7). These results were consistent with the view that polyamines inhibit the induction of AIn, at least in a part, at the level of translation. Very recently it was shown that AIn transcript possesses uORFs with an unsusal translational start codon that is essential for the polyamine-induced repression of the main ORF present at the downstream [48]. Thus, polyamines can regulate the expression of antizyme positively and that of AIn negatively at the translational level, and this may have a profound effect on the antizyme/AIn ratio when polyamine concentrations change. At present we do not know whether polyamines can modulate the localization of antizyme, AIn and ODC during the cell cycle. To this end we need to develop a reliable method to localize polyamines in cells and assess their roles in relation to the function of AIn and antizyme. In conclusion, we showed that AIn protein increased its level in early G1 and G2/M phases of the cell cycle. The first increase appeared to be related to the rapid increase of ODC, a signal to trigger events leading to cell proliferation. On the other hand, experiments using RNA interference suggested that the second increase was associated with promoting the progression of mitosis.

Fig. 7 – Effect of polyamines on the in vitro translation of AIn. The poly (A+) RNA was isolated from HTC cells, and translated in rabbit reticulocyte lysate translation system containing 35 L-[ S]methionine and the indicated concentration of putrescine (A) or spermidine (B). Total protein synthesis was determined by measuring the radioactivity in TCA insoluble fraction. AIn polypeptides in the translation assay mixture were immunoprecipitated by the addition of monoclonal anti-AIn antibody and protein A, separated on SDS-PAGE, and quantified. Details are described in the Materials and methods.

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Acknowledgments This work was supported by Grants from Musashino Gakuin Foundation (to T.O) and (in part) by a Grant from Takeda Science Foundation (to S.M).

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.yexcr.2009.04.024.

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