The essential role of p21 in radiation-induced cell cycle arrest of vascular smooth muscle cell

Journal of Molecular and Cellular Cardiology 37 (2004) 871–880 www.elsevier.com/locate/yjmcc

Original Article

The essential role of p21 in radiation-induced cell cycle arrest of vascular smooth muscle cell Hyo-Soo Kim a,b,1, Hyun-Jai Cho a,b,1, Hyun-Ju Cho a,b, Sun-Jung Park a,b, Kyung-Woo Park a,b, In-Ho Chae a,b, Byung-Hee Oh a,b, Young-Bae Park a,b, Myoung-Mook Lee a,* ,b a

Department of Internal Medicine, Seoul National University College of Medicine, 28 Yongon-dong, Chongno-gu, Seoul 110-744, South Korea b Cardiovascular Laboratory, Clinical Research Institute, Seoul National University Hospital, Seoul, South Korea Received 3 April 2004; received in revised form 2 June 2004; accepted 23 June 2004 Available online 04 August 2004

Abstract The biologic mechanisms for the success and failure of intravascular radiation therapy after angioplasty have not been well studied. We investigated the molecular mechanism of radiation-induced cell cycle arrest in vascular smooth muscle cell (VSMC) and examined whether p21 knock-out is a cause of radiation failure. Using different dosages of gamma radiation, we evaluated the effect of radiation on VSMC apoptosis and cell cycle progression, and its action mechanism. Irradiation significantly retarded the growth of cultured VSMC, which was not due to induction of apoptosis but mainly due to cell cycle arrest. Radiation showed remarkable cell cycle arrest at G1 and G2 phase (G0/G1:S:G2/M phases = 61%:34%:5% with 0 Gy versus 61%:9%:30% with 16 Gy, 12 h after radiation). In immunoblot analysis and kinase assay, radiation increased the expression of p21and decreased the expression and activity of CDK2 and 1. In contrast, radiation did not affect the expression and activity of CDK4 and 6, nor the expression of p27 and p16. When p21 was knocked out, cell cycle of VSMC was not arrested by radiation, leading to increased proliferation. These finding provide the evidence that radiation inhibits VSMC proliferation through cell cycle arrest by enhancing p21 expression and suppressing CDK1 and 2. This observation supports the key role of p21 in radiation-induced cell cycle arrest and the degree of p21 expression may be the possible mechanism of radiation failure and delayed restenosis. © 2004 Elsevier Ltd. All rights reserved. Keywords: Vascular smooth muscle cell; Radiation; Cell cycle; p21; Restenosis

1. Introduction Vascular smooth muscle cell (VSMC) proliferation has long been considered to be a key event in the remodeling process after vascular injury [1,2]. After percutaneous coronary intervention, restenosis continues to be the Achile’s heel. In this aspect, intravascular irradiation after coronary angioplasty had been a promising method of preventing restenosis [3,4]. However, recent studies reporting long term 5 year follow-up data have shown delayed restenosis, which meant there was apparent diminution of efficacy on eliminating the restenotic process over time [5].

* Corresponding author. Tel.: +82-2-760-2226; fax: +82-2-766-8904. E-mail addresses: [email protected] (H.-S. Kim), [email protected] (H.-J. Cho). 1 The first two authors equally contributed to this work. 0022-2828/$ - see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.yjmcc.2004.06.017

The mechanisms regulating the cell cycle in VSMCs remain largely unknown and the effects of radiation on specific cell cycle inhibitors are still unclear. Even less is known about the molecular mechanism of cell growth after radiation injury and the pathogenesis of radiation failure. Expression of p53, a cell cycle transcriptional activator, and p21, a cell cycle inhibitor, have been shown to be elevated after radiation [6]. In addition, p21 is a dual inhibitor of both cyclindependent kinases and proliferating-cell nuclear antigen (PCNA). Furthermore, localized arterial infection with a p21-encoding adenovirus at the time of balloon angioplasty, has been shown to significantly reduce neointimal hyperplasia in animal models [7,8]. Here, we attempted to examine the changes in cyclindependent kinase inhibitors (CDKI) and cell cycle inhibitors after radiation in VSMCs. In addition, we went on further to examine the effects of p21 knock-out on the response of VSMCs to irradiation.

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2. Methods 2.1. Cell culture and irradiation treatment All procedures were approved by the Experimental Animal Committee of Clinical Research Institute, Seoul National University Hospital and complied with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. Cells were given 0, 2, 8, or 16 Gy gamma radiation, using a 137Cs source delivering 3 Gy/min at room temperature, and were harvested 12, 24 and 48 h after irradiation. 2.1.1. Rat VSMC culture VSMCs were isolated from the thoracic aorta of male, 6-week-old Sprague-Dawley rats, by enzyme digestion as previously described [9]. Cells were cultured in DMEM/F12 containing 10% of heat-inactivated fetal bovine serum (GIBCO) and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin). Subconfluent cells at passages 4–7 were used for experiments without synchronization. 2.1.2. Mice VSMC culture VSMCs were isolated from the thoracic aorta of male, BABL/cA mice in the same manner as rat VSMCs. p21–/– cells, a gift from Dr. Kenneth Walsh (Boston University School of Medicine, Boston, MA, USA), were grown in the same media and the p21 knock-out state was reconfirmed by western blot and immunocytochemistry. 2.2. Counting of viable cells and proliferation assay VSMCs were seeded onto 6-well culture plates at a density of 5 × 104 cells per well and grown in DMEM/F12 with 10% FBS. To assess the count of viable cells, an equal volume of trypan blue dye solution (0.1% w/v) was added briefly to stain a portion of the cells. Cells were then loaded into a hemocytometer and counted. The change in the proliferative activity after radiation was evaluated by measuring the incorporation of [3H]thymidine (Amersham) into cellular DNA. Cells were exposed to [3H]thymidine (1 uCi per well) for 24 h, incubated with 5% trichloacetic acid, to precipitate nuclear thymidine, and washed with 100% ethanol. Cells were then lysed with 0.3 N NaOH/2% Na2CO3, and harvested. Radioactivity was counted with a liquid scintillation spectrometer. 2.3. Cell cycle and apoptosis: fluorescence activated cell sorter (FACS) analysis The percentage of VSMCs undergoing apoptosis at each phase of the cell cycle after radiation was estimated by flow cytometry. Briefly, cells were washed with PBS (pH 7.4), and then fixed with ice-cold 70% ethanol at 4 °C for 4 h. The fixed cells were washed with PBS, and incubated for 30 min at room temperature with RNaseA in PBS. Propidium iodide

solution (50 µg/ml) was then added. Flow cytometric analysis was performed on a FACStar Plus (Becton Dickinson) equipped with, LYSIS II version 1.0 and CellFIT Cell-Cycle Analysis version 2.01. 2.4. Western blot analysis Cells were lysed for 1 h at 4 °C in ice-cold lysis buffer. After removal of cellular debris by centrifugation, the supernatant was collected and the protein concentration was analyzed using the Bradford protein assay system (Bio-Rad Lab). Membranes were then incubated with the appropriate primary antibody (dilution 1:500–1000), as follows: CDK1 p34(17), CKD2(M2), CDK4(C-22), CDK6(C-21), p21(C19), p27(F-8), p16(F-12), all from Santa Cruz Biotechnology. The blots were then incubated with horseradish peroxidase-conjugated secondary antibody (ECL, Amersham), and the chemiluminescence was photographed using a CCD camera. The optical density of each band was quantified using image analysis software (TINA 2.0, Raytest). 2.5. CDK activity assays 2.5.1. Immunoprecipitation As has been previously described [10], protein lysates were diluted with RIPA-buffer (50 mM Tris/pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, protease inhibitors) and precleaned with protein A-Sepharose CL-4B beads (50 l; Pharmacia) for 1 h at 4 °C. The beads were then removed by centrifugation. Precleaned lysates were immunoprecipitated by the addition of antibody to 2 µg of CDK1, 2, 4, or 6 and incubated for 2 h at 4 °C. Protein A-Sepharose CL-4B beads were then added and the mixture was gently agitated for an additional 1 h at 4 °C. Immunocomplexes were collected by centrifugation and washed three times with ice-cold RIPA-buffer. 2.5.2. Preparation of retinoblastoma protein (pRb) To prepare the pRb substrate [11] Escherichia coli which had been transformed with pGEX vector containing glutathione-S-transferase retinoblastoma (GST-Rb) fusion gene [10], were grown to saturation overnight, then diluted 1:10 in LB broth containing ampicillin, and incubated at 37 °C for 2 h. The translation of the GST-Rb fusion protein was induced by the addition of 1 mM isopropyl-Dthiogalactopyranoside (IPTG) to the culture for 3 h. The E. coli were pelleted at 4 °C, suspended in PBS, and disrupted by sonication. After centrifugation to remove debris, supernatant fluid was passed through Glutathione Sepharose 4B (Pharmacia) to isolate the GST-Rb fusion protein. It was then cleaved with thrombin to separate the GST and the Rb proteins. Purified Rb proteins were quantified using the Bradford method, and their degree of purity and size were confirmed by electrophoresis on 15% polyacrylamide. 2.5.3. CDK activity assays The purified CDK4 or 6 kinase activity in the immunoprecipitates was measured using Rb protein as substrate and

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CDK1 or 2 activity using histone H1 protein [11,12]. Immunoprecipitated proteins on beads were washed with kinase buffer (50 mM HEPES/pH 7.5, 10 mM MgCl2, 5 mM MnCl2, 1 mM DTT), and then suspended in kinase buffer containing either 2 µg of Rb protein or 2 µg histone H1 (Boehringer, Mannheim), 10 µM ATP, and 10 µCi of [32P]ATP (3000 Ci/mmol; NEN). After incubation for 30 min at 30 °C, with occasional mixing, the reaction was stopped by the addition of a SDS-containing electrophoresis sample buffer. Protein A-immunobeads were boiled for 5 min and then pelleted by centrifugation. Proteins in the reaction mixture were separated by SDS-PAGE. Phosphorylated proteins were visualized by autoradiography of the dried slab gels. The optical density of each band was scanned and quantified using a bio-imaging analyzer (BAS-2500, FUJIFILM). 2.6. Immunocytochemistry Mouse wild type and p21–/– VSMC were cultured at a density of 5 × 104 cells per well onto 6-well plastic slides. Before and 24 h after irradiation, the cells were washed with PBS and fixed in methanol with 3% hydrogen peroxide. Applying normal bovine serum (1:10 in PBS) for 30 min at room temperature blocked non-specific binding. Primary polyclonal anti-human and rat p21 (1:100, Santa Cruz) were allowed to bind overnight at 4 °C [13,14]. After washing the slides in PBS, secondary antibody (DAKO) was applied for 1 h at room temperature and stained with Envision DAB kit (DAKO). The level of non-specific background staining was established for each measurement using cells processed in the same way but without exposure to the primary antibody.

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2.7. Statistical analysis All values are expressed as mean ± S.E.M. of the number of observations. Student’s t-test was used for paired data, and multiple comparisons were performed with the ANOVA method using Bonferroni’s correction for post-hoc analysis. Linear by linear association was used for frequency analysis. All calculations were performed using SPSS version 11.0, and P-value <0.05 was considered statistically significant.

3. Results 3.1. Inhibition of smooth muscle cell growth by irradiation Cells at 70–80% confluence were irradiated with 0, 2, 8 and 16 Gy (n = 9, each) gamma rays. Cell counts at both 24 and 48 h after irradiation was significantly reduced by gamma radiation (Fig. 1). The cell count was 3.28, 2.34, 1.94 and 1.30 × 105 per ml, for 0, 2, 8, and 16 Gy, respectively, at 24 h, and 5.10, 2.00, 1.80 and 1.20 × 105 per ml, respectively, at 48 h. Gamma radiation dose-dependently retarded the cell count increase. As underlying mechanisms of this radiation effect, firstly, we checked the occurrence of apoptosis. Although there were some morphologic features of apoptosis such as cytoplasmic blebbing, nuclear condensation, nuclear fragmentation, and engulfed apoptotic bodies (Fig. 2A–C), the proportion of such cells were minimal (approximately 10 in 1 × 105 cells). In addition, FACS analysis of the VSMCs 12–48 h after irradiation did not show a subdiploid population, which meant the level of apoptosis was negligible.

Fig. 1. Inhibition of VSMC growth by gamma-irradiation Rat VSMC (n = 9) at 80–90% confluence were irradiated with 0, 2, 8, 16 Gy. At 24 h after irradiation, the cell count was 3.28, 2.34, 1.94, 1.30 × 105 per ml, respectively. After 48 h, they measured 5.10, 2.00, 1.80, 1.20 × 105 per ml, respectively. The gamma radiation dose-dependently retarded the cell count increase. Each point is the mean ± S.E.M. * indicates P < 0.05, † P < 0.01 compared with 2, 8 and 16 Gy.

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Fig. 2. Evidence of apoptosis and cell cycle arrest at G1/S and G2/M phases by gamma irradiation At 24 h after 16 Gy radiation, typical morphologic features of apoptosis were observed in rat VSMCs but the degree of apoptosis was minimal. Representative example of apoptotic features, cytoplasmic blebbing and nuclear condensation (A), nuclear fragmentation (B) and engulfed apoptotic body (C). (D) Radiation significantly decreased the cell population in the S phase and increased the cell population in the G2/M phase, which is equivalent to cell cycle arrest in the G1 and G2 phases. P < 0.01 at 12, 24 and 48 h after 16 versus 0 Gy radiation.

3.2. Cell cycle arrest at G1/S and G2/M In generally asynchronized VSMCs without serum fasting preparation, instead of inducing apoptosis, radiation significantly decreased the cell population at the S phase and increased the cell population at the G2/M phase, which is equivalent to cell cycle arrest of the G1 and G2 phases (Fig. 2D). The proportions of cells at G0/G1, S and G2/M phases were 61%, 9% and 30% at 12 h after 16 Gy of radiation (control in the log phase was 61%, 34% and 5%), 65%, 9% and 26% at 24 h (control in the intermediate phase was 70%, 16% and 14%); and 67%, 7% and 26% (control in confluent phase, 78%, 12% and 10%) at 48 h. Such arrest of the cell cycle at the G1 and G2 phases by radiation resulted in a significant reduction of DNA synthesis (by 57%, 24 h after 8 Gy radiation (data not shown). 3.3. Differential effects of gamma radiation on CDKs One of the most important checkpoints in the cell cycle is START in the G1 phase, which is regulated by the D-type

cyclins, CDK4 and 6. Gamma radiation changed neither the protein expression nor the kinase activity (Fig. 3) of both CDK4 and 6 at 12–48 h after irradiation. In contrast to the early G1 CDKs, the protein expression of a late G1 CDK, CDK2, which regulates the G1/S phase transition, was significantly decreased after radiation doses of 8 and 16 Gy (Fig. 3B). The activity of CDK2 was also significantly reduced by radiation (Fig. 4B), which was observed upto 48 h after radiation. The protein expression and activity of CDK1, which regulates the cell cycle at the G2/M, was significantly decreased by 8 and 16 Gy radiation (Figs. 3C and 4C). 3.4. Differential effects of gamma radiation on CDK inhibitors CDKs are under the control of a range of CDK inhibitors, such as p16, p27, and p21, whose clinical implications after angioplasty have been described previously [14]. Expressions of CDK inhibitors in irradiated VSMCs were estimated using western blot analysis (Fig. 5). Among the CDK inhibitors, p21 was significantly enhanced by radiation, whereas

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Fig. 3. Protein expression of CDK6, CDK4, CDK2 and CDK1 after irradiation Western blot analysis was conducted to measure the irradiation-induced changes in the protein expression of the early G1 CDKs, namely CDK6 and CDK4. Results were similar for both 8 and 16 Gy irradiation. The expression of CDK2, an important regulator of G1/S transition, significantly decreased after gamma radiation. The expression of CDK1, an important regulator of the G2/M transition, also significantly decreased after gamma radiation. (A) Representative example of blot. Densitometric data (n = 3) on CDK2 (B) and CDK1 protein expression (C). Each bar represents the mean ± S.E.M. * indicates P < 0.05 compared with 0 Gy, 12 h.

p27, which plays an important role in cell-to-cell contact inhibition, was slightly decreased. The expression of p16, which takes part in regulating only the G1 phase, was negligible and not affected by irradiation. 3.5. Cell cycle arrest and p21 induction in various pre-conditioning We confirmed radiation-induced cell cycle arrest and p21 expression in various conditions for evaluating heterogeneous VSMC population in clinical restenotic lesion. VSMC was prepared with prolonged 48 h-fasting for quiescent G1 synchronized VSMC and PDGF/high dose FBS stimulated VSMC for active proliferating VSMC, in addition to generally asynchronized VSMC (Fig. 2D). In G1 synchronized VSMC (Fig. 6A), radiation-induced cell cycle arrest at G1 phase and high expression of p21 compared with non-radiated VSMC. And in stimulated VSMC for evaluating effect of radiation on G2/M phase and p21 expression (Fig. 6B), we confirmed radiation-induced

cell cycle arrest at G2/M phase and enhanced p21 expression. In addition, sub-G1 population, which indicated apoptosis, was minimal in these various conditions in FACS analysis. 3.6. Effect of radiation on the cell cycle and apoptosis of p21 knock-out VMSCs In order to confirm the essential role of p21 in radiationinduced cell cycle arrest of VSMC, we compared the radiation effect in p21 knock-out VSMC with that in wild type. To ascertain the induction of p21 in wild type, and no expression in p21–/– VSMC after radiation, both immunoblot analysis and immunocytochemistry were performed. The results confirmed increased p21 expression after irradiation in only wild type mouse VSMC but not in p21–/– VSMC (Fig. 7A). Furthermore, the characteristic faint cytoplasmic p21 expression before irradiation and the dense nuclear staining after irradiation in wild type VSMCs were also confirmed (Fig. 7B). When p21 was knocked out, mouse VSMC proliferation was enhanced and radiation-induced cell cycle arrest and apoptosis were not observed, compared with wild type (Fig. 7C, D).

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Fig. 4. Activity of CDK6, CDK4, CDK2 and CDK1 after irradiation in a kinase activity assay The kinase activities of CDK6 and 4 were evaluated by measuring the capacity of phosphorylation of Rb protein in the presence of radioactive ATP. CDK4 and CDK6 activities were not altered by radiation treatment. The kinase activities of CDK2 and 1 were evaluated by measuring the capacity of phosphorylation of histone H1 protein in the presence of radioactive ATP. (A) Radiation significantly decreased the kinase activities of CDK2 and CDK1, compared with the non-irradiated cells. Densitometric data (n = 3) on CDK2 activity (B) and CDK1 activity (C). Each bar represents the mean ± S.E.M. * indicates P < 0.05 compared with 0 Gy, 12 h.

4. Discussion Although it is well known that intracoronary radiation therapy reduces the rate of restenosis through inhibition of VSMC proliferation [3–6], the mechanism underlying the effects of radiation on the cell cycle of VSMCs has not been clarified. In the present study, we showed that gamma radiation inhibits the proliferation and survival of VSMCs in the growth-promoting condition by the induction of G1 and G2 cell cycle arrest rather than significant apoptosis. In addition, the underlying mechanism of radiation-induced cell cycle arrest involved the differential activation of p21 rather than p27 and the differential suppression of CDK1 and 2 rather than CDK4 and 6. Furthermore, the essential role of p21 in radiation-induced cell cycle arrest was confirmed by showing the lack of radiation-induced cell cycle arrest in VSMCs from p21 knock-out mice. 4.1. Biologic responses of VSMC to irradiation The biologic response of cultured VSMCs to radiation has not been extensively investigated. In a previous study, radiation was shown to induce both G1 and G2 arrest in fibro-

blasts, which are biologically similar to VSMCs [15]. However, others have reported that arrest of the G2/M transition was not observed in VSMCs, which were pre-synchronized at the G1 phase prior to beta radiation exposure [6,16]. In the primary and restenotic lesions of human coronary arteries, the proliferative index, assessed by in situ hybridization to PCNA mRNA, was approximately 7% and 20%, respectively [17], which suggests that VSMCs in target lesions for intravascular radiation therapy are not synchronized at the G1 phase. In this study, using asynchronized VSMCs, gamma radiation was found to dose-dependently inhibit VSMC proliferation, and this was due to both G1 and G2 cell cycle arrest rather than induction of significant cellular apoptosis. The difference in VSMC preparation as prolonged 84 h fasting for G1 pre-synchronization before radiation treatment might account for the discrepancy between studies [6]. But, interestingly, in our experiment, radiation-induced cell cycle arrest of the G2/M phase in addition to G1/S occurred in various pre-conditioning, such as G1 synchronized VSMC as 48 h fasting and active cell cycle induced VSMC with PDGF/high dose FBS, as well as generally asynchronized VSMC. In addition, apoptosis occurred minimally in these various conditions.

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Fig. 5. Change of protein expression of CDK inhibitors after irradiation. (A) p21 expression increased significantly after radiation, whereas p27 expression decreased slightly. The expression of p16 was negligible and was not affected by radiation. (B) Densitometric data (n = 3) of relative p21 expression. Each bar represents the mean ± S.E.M. * indicates P < 0.05 compared with 0 Gy, 12 h.

Fig. 6. Evidence of cell cycle arrest and p21 expression in various pre-conditioning In prolonged 48 h fasting (A) and 10 ng/ml PDGF/20% FBS stimulated VSMC (B), after 12 and 24 h 16 Gy irradiation, cell cycle arrest at G1 (A) and G2 (B) phases was observed compared with 0 Gy radiation. And in both conditions, enhanced p21 expression was confirmed by immunoblot. Additionally, sub-G1 portion in FACS analysis, which meant apoptosis, was minimal.

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Fig. 7. Effect of p21 knock-out on radiation-induced cell cycle arrest. When p21was knocked out, mouse VSMC proliferation was enhanced, and radiation-induced cell cycle arrest and apoptosis were not observed in contrast to wild type. The induction of p21 expression after irradiation in wild type but not in p21–/– VSMCs (western blot analysis). (B) Immunocytochemical anlaysis of p21 expression showed characteristic faint cytoplasmic p21 expression before irradiation and dense nuclear staining after irradiation in wild type only. (C) Cell proliferation curves. Each point is the mean ± S.E.M. * P < 0.05 when non-irradiated p21–/– cells were compared with non-irradiated wild type cells; † P < 0.01, 16 Gy irradiated p21–/– cells versus 16 Gy irradiated wild type cells. (D) In wild type VSMC, cell cycle arrest was observed after irradiation, but not in p21–/– cells in FACS analysis.

4.2. Mechanisms of radiation-induced-cell cycle arrest of VSMC The mechanism of G1 and G2 arrest was investigated by the analysis of CDKs (CDK6, 4, 2 and 1) and their inhibitors (p21, p27, and p16) whose roles have been well studied in vasculature [14]. In our study, we found that in terms of the block at the G1/S transition, radiation suppressed the late G1 CDK (CDK2), rather than the early G1 CDKs (CDK4 and 6), showing the differential effects of gamma radiation on the expression of CDKs. The significant suppression of CDK2 may be attributed to the enhanced expression of p21 as shown in the present study. In a previous study using mouse embryonal fibroblast cells [7], radiation-induced cell cycle arrest at the G1/S phase by p21-dependent suppression of CDK2 activity, which was confirmed in p21 knock-out cells. Another inhibitor of CDK2 is p27, which is similar to p21 in terms of its peptide sequence and function as a general inhibitor of CDKs [18]. In our study, p27 expression did not increase but rather decreased slightly after irradiation, which suggests that p27 does not contribute significantly to radiation-induced G1/S cell cycle arrest. p27 levels are

known to be increased by transforming growth factor-b (TGF-b) treatment or by cell-to-cell contact [18,19]. In addition, investigators reported that endovascular irradiation inhibits the expression of active TGF-b1 in arteries following balloon injury [20]. Taken together, we believe that the radiation-induced p27 decrease observed in the present study, may be due to decreased TGF-b expression or decreased cell-to-cell contact resulting from retarded growth of cultured cells after irradiation. Although both the late G1 CDK (CDK2) and the early CDKs (CDK4 and 6) are under the influence of p21, only the suppression of CDK2 was observed in the present study. The differential effect of radiation and increased p21 expression on the CDKs is unclear. However, we could postulate some possible explanations. One may be the negligible expression of p16, a specific inhibitor of CDK4 and 6, in cultured VSMCs as shown in this study, as well as in normal and atherosclerotic arteries [14]. The level of p16 is known to increase in cells lacking functional pRb [21,22]. Therefore, in VSMCs with functional pRb, p16 expression may be too low to inhibit the activities of CDK4 and 6. Another possible

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explanation is the difference in binding affinities of CDK2 and 4 to their inhibitors such as p21 or p27. In a previous study using fibroblasts, investigators showed that p21 alone is likely to be sufficient for the inhibition of CDK2 whereas the activation of both p21 and p27 is necessary to inhibit CDK4 [23]. In terms of G2/M cell cycle arrest, radiation-induced inhibition of CDK1 may result in cell cycle arrest at the G2/M phase [24–26]. A previous study [27] suggested that p21 is responsible for regulating the G2/M checkpoint, blocking the entrance into the M phase in the presence of chromosomal damage, and preventing cells from apoptotic death due to aberrant cell cycle progression. In several mammalian cells other than VSMCs, DNA damage by ionizing radiation or anticancer drugs is sensed by ataxia telangiectasia mutant (ATM) gene, which through several steps leads to inhibition of CDK1. In the present study, radiation significantly inhibited the expression and the activity of CDK1 and arrested cells at the G2/M phase. This may be related to the enhanced expression of p21. These responses of VSMCs to radiation, i.e. enhanced p21 expression and cell cycle arrest both at the G1/S and G2/M phase, could provide time for repair of damaged DNA and allow cells to avoid apoptotic death. To confirm the essential role of p21 in radiation-induced cell cycle arrest, VSMCs from p21 knock-out mice were used. When p21 was knocked out, VSMC proliferation was enhanced in ordinary culture condition. In addition, radiation-induced cell cycle arrest and apoptosis were not observed in contrast with wild type cells, confirming the essential role of p21 in radiation-induced cell cycle arrest in VSMCs.

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induction has not been initiated, cannot be prevented. Intravascular irradiation, however, can induce p21 at an earlier period to block the proliferative surge, which may be why radiation is effective at preventing restenosis.

5. Conclusion Our results show that the major effect of gamma radiation on inhibition of restenosis is through G1 and G2 cell cycle arrest rather than through induction of apoptosis, which is a result of a differential inhibition and induction of CDKs and its inhibitors. Furthermore, the induction of p21 appears to play a key role in radiation-induced cell cycle arrest, suggesting that the expansion of p21 deficient VSMCs may be a possible mechanism of radiation failure and delayed catch-up restenosis. Considering that radiation-induced p21 but not p27, adjunctive therapy to induce p27 expression at the irradiated site may be helpful to overcome the limitation of intravascular radiation therapy, further studies will be necessary to confirm this hypothesis.

Acknowledgments This study was supported by a grant from the Korea Health 21 R&D project, Ministry of Health & Welfare (02PJ10-PG8-EC01-0026), and a grant from Stem Cell Research Center, Republic of Korea (SC13122). Dr. Hyo-Soo Kim is an investigator of the Aging and Apoptosis Research Center of Seoul National University sponsored by KOSEF.

4.3. Clinical implications The results of this study suggest the mechanisms by which intravascular radiation reduces the incidence of restenosis. Recently, the sequential changes of cell death and proliferation and the role of CDK inhibitors after balloon injury has been well described [14,28]. Firstly within 24 h of balloon injury, massive cell death due to mechanical injury occurs in the media [29], which results in a rapid decrease of cell density in the media and a reduction of p27 expression, which is dependent on cell density. Hence, during the window period of several days after injury, when the inhibition of the cell cycle by p27 is absent and increased p21 expression has not had time to develop, explosive cell growth occurs. Finally, when the repair process is completed, not only does p27 expression increase to its baseline level as cell density increases, but p21 is also induced to help prevent cell growth. In terms of apoptosis, vascular injury led to massive apoptosis in early period, but there were no quantitative differences in the amount of apoptotic cells between irradiated and control-injured vessel in the previous studies [6,30]. Therefore, these findings imply that the induction of apoptosis may not be the main mechanism of radiation benefit. Without intravascular irradiation, the proliferative surge during the window period [14] when p27 decreases but p21

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