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p21Waf1 pathway in glioma cell
BBRC Biochemical and Biophysical Research Communications 317 (2004) 902–908 www.elsevier.com/locate/ybbrc
Overexpression of RFT induces G1–S arrest and apoptosis via p53/p21Waf1 pathway in glioma cellq Hideyuki Kano,a Yoshiki Arakawa,a,b Jun A. Takahashi,a Kazuhiko Nozaki,a Yasuhiro Kawabata,a Kenji Takatsuka,d Ryoichiro Kageyama,d Tetsuya Ueba,a,c,* and Nobuo Hashimotoa,* a b
Department of Neurosurgery, Kyoto University Graduate School of Medicine, Sakyo-ku, Kyoto 606-8315, Japan Department of Pharmacology, Kyoto University Graduate School of Medicine, Sakyo-ku, Kyoto 606-8315, Japan c Department of Neurosurgery, Kishiwada City Hospital, Osaka 596-8501, Japan d Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan Received 1 March 2004
Abstract The regulator of fibroblast growth factor 2 (FGF-2) transcription (RFT) has been reported to be a transcriptional repressor of FGF-2 and induce glioma cell death by its overexpression. Here we report that RFT regulates cell cycle as well as apoptosis by a novel mechanism. RFT expressed in some glioma cell lines, U138MG and T98G, but neither in U87MG nor U251MG. Overexpressed RFT-induced apoptosis in U87MG and U138MG with functioning-type p53 but neither in U251MG nor T98G with nonfunctioning-type p53. Administration of FGF-2 failed to prevent RFT-induced apoptosis. Overexpression of RFT caused G1–S arrest and upregulated both the phosphorylation of p53 at Ser-15 and the expression level of p21Waf1 . Furthermore, RNAi knockdown of p53 abolished RFT-induced apoptosis in U87MG. Taken together, our results support that RFT regulates G1–S transition and apoptosis via p53/p21Waf1 pathway. Ó 2004 Elsevier Inc. All rights reserved. Keywords: RFT; FGF-2; p53; p21Waf1 ; Cell cycle; Apoptosis
The regulator of fibroblast growth factor 2 (FGF-2) transcription (RFT) was identified by a yeast one-hybrid screening with a defined motif in FGF-2 promoter as a target sequence. The target sequence was tandemly repeated gccgaac at both sides of the transcription initiation site of the human FGF-2 gene. In vitro assay, RFT acted as a repressor of FGF-2 expression at the transcriptional level. Two splice variants, RFT-A0 and RFT-
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Abbreviations: RFT, the regulator of fibroblast growth factor 2 transcription; FGF-2, fibroblast growth factor 2; MBD1, methyl-CpGbinding protein 1; GFP, green fluorescent protein; FACS, fluorescence activated cell sorting; BrdU, bromodeoxyuridine; siRNA, small interfering RNA; PIK, phosphoinositide-3 kinase-related kinase; ATM, ataxia telangiectasia mutated. * Corresponding authors. Fax: +81-75-751-4693 (N. Hashimoto). E-mail addresses: [email protected] (T. Ueba), [email protected] (N. Hashimoto). 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.03.120
B, were also identified and they had deletions in the putative DNA-binding domain and failed to bind FGF2 promoter and repress FGF-2 gene expression [28]. RFT contains methyl-CpG-binding domain (MBD domain), which is characteristic of MBD protein family [4]. Overexpression of RFT-induced U87MG glioma cell death and the mechanism was thought to be that RFT directly decreased the expression level of FGF-2 by repressing the transcriptional activity [28]. However, we found that overexpression of RFT did not induce cell death in some glioma cell lines and that FGF-2 was not essential for RFT-induced apoptosis. We have been attempting to explore other possible mechanisms of RFT-induced glioma cell death. It has been widely accepted that the p53 tumor suppressor protein is ubiquitously involved in growth arrest and/or apoptosis of the most cells, and that it regulates the expression level of p21Waf1 , which inhibits the
H. Kano et al. / Biochemical and Biophysical Research Communications 317 (2004) 902–908
cyclin-dependent kinases [14,17,20]. For mediating the p53-dependent apoptotic pathway, several upregulation of p53 target genes, including Bax and PIG, have been reported [18,21]. It has been shown that phosphorylation and acetylation of p53 play important roles for regulation of its biological activities [8,22]. Indeed, phosphorylation of p53 at Ser-15 and Ser-20 has been shown to be involved in activation of p53 [6,24–26]. Moreover, induction of wild-type p53 causes cell cycle control and apoptosis in several p53-mutated cancer cells [17]. However, It remained unclear whether p53 take part in RFT-induced cell death. Here we report that G1–S arrest and apoptosis caused by RFT were associated with the expression of functioning-type p53 in glioma cells and need the activation of p53/p21Waf1 pathway.
Materials and methods Construction and infection of retrovirus. For construction of CLIG His-tagged RFT, cDNAs for His-tagged RFT were cloned into the EcoRI site of pCLIG, which directs expression of the cloned genes together with GFP from the upstream long-terminal repeat (LTR) promoter. Retroviral vectors and pVSV-G were cotransfected with Lipofectamine 2000 (Gibco-BRL) into 293GP, a cell line which expresses gag and pol. The supernatant was collected 2 days later and concentrated by 100-fold concentration with Centricon Plus-20 (Amicon, Beverly, MA), as described previously [11]. The viral titer determined by GFP positive cells was 1 106 colony-forming units/ml. To viral medium was added 1/50 volume of fresh medium in each well. And we confirmed the efficiency of infection was 80% and more in each cell lines. Cell lines and cultures. The human glioma cell line U87MG, U138MG, U251MG, and T98G cells were provided from ATCC and cultured in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum (BioWhittaker) in a humidified atmosphere with 5% CO2 . RNA and RT-PCR. Total RNA was isolated from the cells using Trizol (Invitrogen) according to the manufacturer’s instructions. Reverse transcription (RT) was performed with a first-strand cDNA kit following the protocol recommended by the manufacturer (Life Technologies). RT-PCR exponential phase was determined on 25–35 cycles to allow semiquantitative comparisons among cDNAs developed from identical reactions. Each PCR regime involved a 2 min initial denaturation step at 94 °C, followed by 35 cycles (for RFT), 25 cycles (for G3PDH) at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min. The sequences of RFT primers were 50 -CCCAACTGGTAAACATGCCC-30 (a forward primer) and 50 -GCCGCTTCATGGCAAACT-30 (a reverse primer). The sequence of G3PDH primers were 50 -aagaaggtggtgaagcaggcgtcgg-30 (a forward primer) and 50 -aggccatgtggccatgaggtcc-30 (a reverse primer). Cell proliferation assay. Aliquots of cell suspension containing 104 cells were inoculated into each well of 96-well tissue culture microplates. After the cells attached to the substratum, the medium was replaced with fresh medium containing 1 ll virus solution. FGF-2 (PEPRO Tech EC) was dissolved directly in medium. The cells were incubated for 24, 48, and 72 h and then 20 ll of MTS solution (Qiagen) added to the wells and the plates were further incubated for 1 h at 37 °C. Absorbance at 490 nm was read by an automicroplate reader. Each experiment was performed at least three times in triplicate.
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TdT-mediated dUTP-X nick end-labeling. TdT-mediated dUTP-X nick end-labeling (TUNEL) staining was performed as described in the manufacturer’s protocol (in situ apoptosis detection kit, Takara). Cell cycle analysis. Cells were trypsinized, washed in PBS, collected, and fixed with 70% ethanol. After storage for at least 3 h at )20 °C, cells were washed with PBS and resuspended in PBS containing RNAase A (0.2 lg/ml) and propidium iodide (20 lg/ml, Sigma). The stained cells were analyzed on a Becton–Dickinson FACScan flow cytometer. Immunofluorescence microscopy. Immunocytochemistry was performed essentially as described [2], using a Carl Zeiss LSM 510 system. Primary and secondary antibodies were as follows: rabbit polyclonal anti-GFP antibody (MBL), anti-6 His monoclonal antibody (BD Biosciences), rat anti-tubulin monoclonal antibody (Chemicon), and Alexa Fluor 488-, 594-, 633-conjugated goat anti-rabbit, and antimouse secondary (Molecular Probes). TO-PRO3 iodide (Molecular Probes) was used for nuclear staining. In all image analyses, no background subtraction was carried out, and all pseudocolor representations were assembled using Photoshop Ver 7.0 (Adobe) for illustrative purpose only. BrdU incorporation assay. The proportion of cells in S phase of the cell cycle was determined through immunocytochemistry for incorporated BrdU. Cells were plated onto collagen, type I coated BIOCOAT Culture Slide (Becton–Dickinson Labware), and treated for 24 h after infection. Cells were incubated with 3 lg/ml BrdU in fresh DMEM at 37 °C for 12 h, prior to fixation with cold 4% parafolmaldehide/PBS. DNA in the fixed cells was denatured by incubating slides in 1.5 N HCl and 300 lg/ml RNase after 30 min at 37 °C. A monoclonal mouse antiBrdU antibody (Boehringer–Mannheim) was used at 1:20 dilution. Nuclei were counter-stained with TO-PRO3. The fraction of nuclei staining positive for BrdU was determined at high power. At least five fields were counted for each well, and all experiments were performed in triplicate. Groups were compared using factorial analysis of variance (ANOVA), and P values were obtained following application of Fisher’s protected least significant difference post hoc procedure to the data. A P value <0.05 following this procedure is considered significant. These analyses were completed using Prism 3.0 for the Macintosh. Western blot analysis. Western blot analysis was performed essentially as described [2]. Briefly, cells were collected 24, 48, and 72 h after infection, lysed in the lysis buffer. The mixtures were boiled for 5 min and subjected to SDS–PAGE. Antibodies against p21Waf1 (Santa Cruz), anti-b-tubulin monoclonal antibody (TUB.2.1) (Sigma–Aldrich), 6 His (BD Biosciences), p53, p53 phosphorylated at Ser15 (Cell Signaling) were purchased. Protein knockdown of p53 by RNA interference using siRNA. p53 pub siRNA and Control (non-silencing) siRNA were purchased from Qiagen. Cotransfection of expression vector (pCLIG-RFT, pCLIG) and siRNA (p53 pub. siRNA, scramble siRNA) for U87MG cells (1 104 cells/ml) was performed with Lipofectamine 2000 in 6-well plates for Western blot. Per well, 4 ml lipofectamine 2000 diluted in 100 ml Opti-MEM was applied to a premix consisting of siRNA (20 mM, 4 ml)/Opti-MEM (100 ml), and incubated for 30 min. The whole mixture was added to the medium, which then was changed to DMEM 3 h after transfection. Cells were incubated for 24–48 h before analysis of knockdown p53 by Western blot as described above. For immunocytochemistry, cotransfection of expression vector (pCLIG-RFT, pCLIG) and siRNA to U87MG cells (5 105 cells/ml) were performed with Lipofectamine 2000 in 8-well culture slides. Briefly, for 1 105 cells, 2 ml Lipofectamine 2000 diluted in 30 ml OptiMEM was applied to a pCLIG-RFT or pCLIG (1 lg)/siRNA (20 lM, 2 ll)/Opti-MEM (30 ll) mixture, and incubated for 30 min. The entire mixture was added to the cell-containing medium, which then was changed to DMEM 3 h after transfection. Cells were incubated for 24 h before immunocytochemistry were performed as described above and counting of His positive cells.
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Results
RFT induces G1–S arrest preceding apoptosis
Overexpression of RFT induces apoptosis in glioma cell
We next tested whether overexpression of RFT influence cell cycle. Cell cycle was examined by fluorescence activated cell sorting (FACS) analysis. The FACS analysis demonstrated that overexpression of RFT blocked G1–S progression in U87MG (Fig. 3A) but not in U251MG (data not shown). To determine the effect of G1–S progression, we performed bromodeoxyuridine (BrdU) incorporation assay in 24–36 h after the retrovirus infection. Overexpression of RFT disturbed BrdUincorporation in U87MG (Fig. 2B) and U138MG, but neither U251, T98G nor HeLa cell (data not shown). Cell death was examined by TUNEL assay. TUNEL positive cells were increasing in RFT-expressed U87MG and U138MG in time-dependent manner after retrovirus infection (Fig. 4). However, TUNEL positive cells did neither increase in U251MG nor T98G after overexpression of RFT (data not shown). These results suggested that RFT could induce apoptosis following G1–S arrest in glioma cell.
To test whether RFT induces apoptosis in various glioma cell lines, we overexpressed RFT in glioma cell lines, U87MG, U138MG, U251MG, and T98G, using a replication-incompetent retrovirus CLIG system [11]. Expressions of His-tagged RFT and GFP were simultaneously confirmed 24 h after retrovirus infection to all glioma cell lines. Immunofluorescence study demonstrated that RFT was localized in the nucleus of all cell lines (Fig. 1). In cell growth assay using MTS, overexpression of RFT-induced cell death in U87MG and U138MG 48 h after retrovirus infection but neither in U251MG, T98G (Fig. 2A) nor HeLa cell (data not shown), although the infection efficiencies were the same in all cell lines. Previous report demonstrated that the reduction of FGF-2 caused apoptosis of RFTexpressed U87MG. Therefore, we evaluated whether FGF-2 took a part of apoptosis induced by RFT. However, the addition of FGF-2 by various concentrations did not rescue apoptosis in RFT-expressed U87MG nor U138MG (Fig. 2A). We investigated whether there were any differences of RFT expression in each glioma cell lines. We found that RFT protein expressed in U138MG and T98G but neither in U87MG nor U251MG by RT-PCR using the specific exon of RFT (Fig. 2B). These results indicated that RFT-induced apoptosis was not artificial phenomenon by its overexpression, and that it was not directly caused by the FGF-2 reduction.
RFT phosphorylates p53 and increases expression of p21Waf1 We tried to find the mechanism of RFT regulation in G1–S arrest and apoptosis. The p53 protein is ubiquitously involved in G1–S arrest and/or apoptosis of the most cells, mainly through the control of p21Waf1 expression level [14,17,20]. It has been shown that U251MG and T98G have mutated non-functioningtype p53, but U87MG and U138MG have functioning-
Fig. 1. Localization of overexpressed RFT in glioma cell lines. Simultaneous expressions of RFT and GFP were confirmed 24 h after virus infection to glioma cell lines. By the detection of His-tagged RFT, the localization of RFT was in the nucleus of all cell lines. Scale bars, 20 lm.
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Fig. 2. Effect of cell growth by RFT and its differential expression level in each glioma cell lines. (A) Overexpression of RFT by retrovirus CLIG-HisRFT inhibited cell growth and induced cell death in U87MG and U138MG, but neither U251MG nor T98G 48 h after infection. The addition of FGF-2 (1 lg/ml) did not rescue the obstacle of cell growth in RFT expressed U87MG nor U138MG. % control; RFT overexpressed cells/control cells X100 (B) RFT highly expressed in some glioma cell lines, U138MG and T98G, but neither U87MG nor U251MG.
Fig. 3. G1–S arrest and apoptosis induced by overexpression of RFT. (A) FACS analysis demonstrates that RFT prevented G1–S progression in U87MG 48 h after the infection. (B) RFT downregulated BrdU incorporations in U87MG cell but not U251. ***p < 0:001; scale bars, 50 lm.
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transient expression and knockdown method that U87MG cell was introduced the expression vector CLIG-RFT and small interfering RNA (siRNA) of p53 by using lipofection method, previously described method [2]. The experiment revealed that RNAi knockdown of p53 abolished RFT-induced apoptosis in U87MG cell (Fig. 6A). Moreover, RNAi knockdown of p53 inhibited the increasing expression of p21Waf1 (Fig. 6B). Our findings indicated that RFT regulates G1–S transition and apoptosis via the activation of p53/ p21Waf1 pathway, although it was still unclear how RFT activated p53/p21Waf1 pathway.
Discussion
Fig. 4. Detection of apoptosis induced by RFT. TUNEL staining indicate that TUNEL positive cells were increasing in RFT-expressed U87MG and U138MG in a time-dependent manner but control virus did not, whereas only a few TUNEL positive cells were seen at 24 h in both infected cells. Scale bars, 50 lm.
type p53. The p53 reporter activities were reported to be 0, 0, 36, and 9 in U251MG, TG98, U87MG, and U138MG, respectively [12,23,29]. Therefore, we tested the expression and phosphorylation levels of p53 in the RFT regulation of G1–S arrest and apoptosis in U87MG with wild-type p53. We found that the expression levels of p53 were increased and its phosphorylation at Ser-15 residue was highly increased in RFT-expressed U87MG. Moreover, the expression level of p21Waf1 was also increased (Fig. 5). We evaluated whether p53 pathway was essential for RFT regulation of cell cycle and apoptosis. We used the
Fig. 5. Activation of p53 p21Waf1 pathway by RFT. Overexpression of RFT upregulated the expression level and the phosphorylation of p53 at Ser-15 residue. Moreover, RFT increased the expression level of p21Waf1 in U87MG.
In the previous report, RFT decreased FGF-2 promotor activity in vitro and it was identified as a repressor of FGF-2 transcription. Overexpression of RFT using adeno-associated virus led glioma cell to FGF-2 reduction and apoptosis [28]. It has been widely accepted that FGF-2 signaling is important for survival of most types of cell, expressing both the FGF-2 and the FGF-2 receptor genes [1,9]. A neutralizing antibody against basic FGF-2 or blocking peptides of FGF-2 receptor has been reported to inhibit the growth of U87MG, U251MG, T98G, and HeLa cells [15,19,27]. However, we found that RFT-induced apoptosis was not rescued by the addition of FGF-2 in U87MG and U138MG, and that overexpression of RFT did not induce apoptosis in U251MG, T98G nor HeLa cell. High expression of endogenous full-length RFT was identified in U138MG and T98G. These indicated that RFT could not always suppress the expression of FGF-2 in vivo and that unknown mechanisms might activate RFT to repress the FGF-2 transcription. The tumor suppressor protein p53 is known as ubiquitous regulator of cell cycle and/or apoptosis. In glioma cell lines, U251MG and T98G have mutatedtype p53, which is known to be loss of function, but U87MG and U138MG has functioning-type p53 (wildtype p53 in U87MG, mutated type in U138MG) [12,29]. In this study, RFT regulated cell cycle through the activation of p53 by phosphorylation at ser-15 and the upregulation of p21Waf1 expression level, and the knockdown of p53 prevented apoptosis in RFT-overexpressed U87MG. The p53 is essential for the regulation of cell cycle and apoptosis by RFT. Therefore, overexpression of exogenous RFT did not induce apoptosis in U251MG nor T98G with non-functioningtype p53. It is still unclear how RFT activate p53 pathway. RFT might indirectly activate p53 by a transcription of some genes, which are involved in activation of p53, or it could activate directly. Recent studies has shown that members of the phosphoinositide-3 kinase-
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Fig. 6. RNAi knockdown of p53 abolished RFT-induced apoptosis in U87MG (A,B). By the transient expression and knockdown method, U87MG was introduced the both of CLIG-RFT and siRNA-p53. RNAi knockdown of p53 abolished RFT-induced apoptosis in U87MG 48 h after the transfection. (C) RNAi knockdown of p53 inhibited the expression of p21Waf1 as well as that of p53. ***p < 0:001; **p < 0:025; scale bars, 50 lm.
related kinase (PIK) superfamily, including the TOR subfamily, the ataxia telangiectasia mutated (ATM) subfamily, and DNA-PK, regulate cell-cycle progression and DNA repair in eukaryotic cells. ATM and ATR participate in the activation of cell cycle checkpoints induced by DNA damage [13,30]. ATM has been shown to be involved in the DNA damage-induced phosphorylation of p53 at Ser-15 [3,5]. Ser-15 is a functionally important residue within the p53 amino-terminal region [7], and phosphorylation of Ser-15 represents an early cellular response to a variety of genotoxic stresses [24,26]. The p53 phosphorylated at Ser-15 displayed reduced binding to the inhibitor protein MDM2 [25]. Because the association with MDM2 targets p53 for proteosome-mediated degradation and inhibits its transactivating function [10,16], Ser-15 phosphorylation promotes both the accumulation and functional activation of p53 in response to DNA damage. Two RFT spliced variants with deletions in the putative DNA-binding domain failed to induce G1–S arrest and apoptosis. RFT need to bind DNA with DNA-binding domain when it function. Therefore, there might be a link between RFT and PIK family involving the sense of DNA damage and the regulation of G1–S transition. In conclusion, RFT regulates cell cycle and apoptosis in p53-dependent manner. For these regulations, RFT phosphorylates p53 at Ser-15 and upregulates the expression level of p21Waf1 . Although its mechanism is not still unknown, the specific exon of RFT might act on the activation of p53 pathway. Our results are suggested that RFT could not only function for the FGF-2 repressor but also the maintenance of genome stability through p53/p21Waf1 pathway.
Acknowledgments The authors thank Furuyashiki T, Thumkeo D, and Yamana N (Dept. of Pharmacology, Kyoto University) for discussion, Dr. Kafri T (Univ. North Carolina) for pcDNA-VSV-G. This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (N.H., K.N., J.T., and R.K.) and the Ministry of Health, Labour and Welfare (N.H.). Y.A. is a postdoctoral fellow from the Japan Society for Promotion of Science.
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Overexpression of RFT induces G1–S arrest and apoptosis via p53/p21Waf1 pathway in glioma cellq Hideyuki Kano,a Yoshiki Arakawa,a,b Jun A. Takahashi,a Kazuhiko Nozaki,a Yasuhiro Kawabata,a Kenji Takatsuka,d Ryoichiro Kageyama,d Tetsuya Ueba,a,c,* and Nobuo Hashimotoa,* a b
Department of Neurosurgery, Kyoto University Graduate School of Medicine, Sakyo-ku, Kyoto 606-8315, Japan Department of Pharmacology, Kyoto University Graduate School of Medicine, Sakyo-ku, Kyoto 606-8315, Japan c Department of Neurosurgery, Kishiwada City Hospital, Osaka 596-8501, Japan d Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan Received 1 March 2004
Abstract The regulator of fibroblast growth factor 2 (FGF-2) transcription (RFT) has been reported to be a transcriptional repressor of FGF-2 and induce glioma cell death by its overexpression. Here we report that RFT regulates cell cycle as well as apoptosis by a novel mechanism. RFT expressed in some glioma cell lines, U138MG and T98G, but neither in U87MG nor U251MG. Overexpressed RFT-induced apoptosis in U87MG and U138MG with functioning-type p53 but neither in U251MG nor T98G with nonfunctioning-type p53. Administration of FGF-2 failed to prevent RFT-induced apoptosis. Overexpression of RFT caused G1–S arrest and upregulated both the phosphorylation of p53 at Ser-15 and the expression level of p21Waf1 . Furthermore, RNAi knockdown of p53 abolished RFT-induced apoptosis in U87MG. Taken together, our results support that RFT regulates G1–S transition and apoptosis via p53/p21Waf1 pathway. Ó 2004 Elsevier Inc. All rights reserved. Keywords: RFT; FGF-2; p53; p21Waf1 ; Cell cycle; Apoptosis
The regulator of fibroblast growth factor 2 (FGF-2) transcription (RFT) was identified by a yeast one-hybrid screening with a defined motif in FGF-2 promoter as a target sequence. The target sequence was tandemly repeated gccgaac at both sides of the transcription initiation site of the human FGF-2 gene. In vitro assay, RFT acted as a repressor of FGF-2 expression at the transcriptional level. Two splice variants, RFT-A0 and RFT-
q
Abbreviations: RFT, the regulator of fibroblast growth factor 2 transcription; FGF-2, fibroblast growth factor 2; MBD1, methyl-CpGbinding protein 1; GFP, green fluorescent protein; FACS, fluorescence activated cell sorting; BrdU, bromodeoxyuridine; siRNA, small interfering RNA; PIK, phosphoinositide-3 kinase-related kinase; ATM, ataxia telangiectasia mutated. * Corresponding authors. Fax: +81-75-751-4693 (N. Hashimoto). E-mail addresses: [email protected] (T. Ueba), [email protected] (N. Hashimoto). 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.03.120
B, were also identified and they had deletions in the putative DNA-binding domain and failed to bind FGF2 promoter and repress FGF-2 gene expression [28]. RFT contains methyl-CpG-binding domain (MBD domain), which is characteristic of MBD protein family [4]. Overexpression of RFT-induced U87MG glioma cell death and the mechanism was thought to be that RFT directly decreased the expression level of FGF-2 by repressing the transcriptional activity [28]. However, we found that overexpression of RFT did not induce cell death in some glioma cell lines and that FGF-2 was not essential for RFT-induced apoptosis. We have been attempting to explore other possible mechanisms of RFT-induced glioma cell death. It has been widely accepted that the p53 tumor suppressor protein is ubiquitously involved in growth arrest and/or apoptosis of the most cells, and that it regulates the expression level of p21Waf1 , which inhibits the
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cyclin-dependent kinases [14,17,20]. For mediating the p53-dependent apoptotic pathway, several upregulation of p53 target genes, including Bax and PIG, have been reported [18,21]. It has been shown that phosphorylation and acetylation of p53 play important roles for regulation of its biological activities [8,22]. Indeed, phosphorylation of p53 at Ser-15 and Ser-20 has been shown to be involved in activation of p53 [6,24–26]. Moreover, induction of wild-type p53 causes cell cycle control and apoptosis in several p53-mutated cancer cells [17]. However, It remained unclear whether p53 take part in RFT-induced cell death. Here we report that G1–S arrest and apoptosis caused by RFT were associated with the expression of functioning-type p53 in glioma cells and need the activation of p53/p21Waf1 pathway.
Materials and methods Construction and infection of retrovirus. For construction of CLIG His-tagged RFT, cDNAs for His-tagged RFT were cloned into the EcoRI site of pCLIG, which directs expression of the cloned genes together with GFP from the upstream long-terminal repeat (LTR) promoter. Retroviral vectors and pVSV-G were cotransfected with Lipofectamine 2000 (Gibco-BRL) into 293GP, a cell line which expresses gag and pol. The supernatant was collected 2 days later and concentrated by 100-fold concentration with Centricon Plus-20 (Amicon, Beverly, MA), as described previously [11]. The viral titer determined by GFP positive cells was 1 106 colony-forming units/ml. To viral medium was added 1/50 volume of fresh medium in each well. And we confirmed the efficiency of infection was 80% and more in each cell lines. Cell lines and cultures. The human glioma cell line U87MG, U138MG, U251MG, and T98G cells were provided from ATCC and cultured in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum (BioWhittaker) in a humidified atmosphere with 5% CO2 . RNA and RT-PCR. Total RNA was isolated from the cells using Trizol (Invitrogen) according to the manufacturer’s instructions. Reverse transcription (RT) was performed with a first-strand cDNA kit following the protocol recommended by the manufacturer (Life Technologies). RT-PCR exponential phase was determined on 25–35 cycles to allow semiquantitative comparisons among cDNAs developed from identical reactions. Each PCR regime involved a 2 min initial denaturation step at 94 °C, followed by 35 cycles (for RFT), 25 cycles (for G3PDH) at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min. The sequences of RFT primers were 50 -CCCAACTGGTAAACATGCCC-30 (a forward primer) and 50 -GCCGCTTCATGGCAAACT-30 (a reverse primer). The sequence of G3PDH primers were 50 -aagaaggtggtgaagcaggcgtcgg-30 (a forward primer) and 50 -aggccatgtggccatgaggtcc-30 (a reverse primer). Cell proliferation assay. Aliquots of cell suspension containing 104 cells were inoculated into each well of 96-well tissue culture microplates. After the cells attached to the substratum, the medium was replaced with fresh medium containing 1 ll virus solution. FGF-2 (PEPRO Tech EC) was dissolved directly in medium. The cells were incubated for 24, 48, and 72 h and then 20 ll of MTS solution (Qiagen) added to the wells and the plates were further incubated for 1 h at 37 °C. Absorbance at 490 nm was read by an automicroplate reader. Each experiment was performed at least three times in triplicate.
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TdT-mediated dUTP-X nick end-labeling. TdT-mediated dUTP-X nick end-labeling (TUNEL) staining was performed as described in the manufacturer’s protocol (in situ apoptosis detection kit, Takara). Cell cycle analysis. Cells were trypsinized, washed in PBS, collected, and fixed with 70% ethanol. After storage for at least 3 h at )20 °C, cells were washed with PBS and resuspended in PBS containing RNAase A (0.2 lg/ml) and propidium iodide (20 lg/ml, Sigma). The stained cells were analyzed on a Becton–Dickinson FACScan flow cytometer. Immunofluorescence microscopy. Immunocytochemistry was performed essentially as described [2], using a Carl Zeiss LSM 510 system. Primary and secondary antibodies were as follows: rabbit polyclonal anti-GFP antibody (MBL), anti-6 His monoclonal antibody (BD Biosciences), rat anti-tubulin monoclonal antibody (Chemicon), and Alexa Fluor 488-, 594-, 633-conjugated goat anti-rabbit, and antimouse secondary (Molecular Probes). TO-PRO3 iodide (Molecular Probes) was used for nuclear staining. In all image analyses, no background subtraction was carried out, and all pseudocolor representations were assembled using Photoshop Ver 7.0 (Adobe) for illustrative purpose only. BrdU incorporation assay. The proportion of cells in S phase of the cell cycle was determined through immunocytochemistry for incorporated BrdU. Cells were plated onto collagen, type I coated BIOCOAT Culture Slide (Becton–Dickinson Labware), and treated for 24 h after infection. Cells were incubated with 3 lg/ml BrdU in fresh DMEM at 37 °C for 12 h, prior to fixation with cold 4% parafolmaldehide/PBS. DNA in the fixed cells was denatured by incubating slides in 1.5 N HCl and 300 lg/ml RNase after 30 min at 37 °C. A monoclonal mouse antiBrdU antibody (Boehringer–Mannheim) was used at 1:20 dilution. Nuclei were counter-stained with TO-PRO3. The fraction of nuclei staining positive for BrdU was determined at high power. At least five fields were counted for each well, and all experiments were performed in triplicate. Groups were compared using factorial analysis of variance (ANOVA), and P values were obtained following application of Fisher’s protected least significant difference post hoc procedure to the data. A P value <0.05 following this procedure is considered significant. These analyses were completed using Prism 3.0 for the Macintosh. Western blot analysis. Western blot analysis was performed essentially as described [2]. Briefly, cells were collected 24, 48, and 72 h after infection, lysed in the lysis buffer. The mixtures were boiled for 5 min and subjected to SDS–PAGE. Antibodies against p21Waf1 (Santa Cruz), anti-b-tubulin monoclonal antibody (TUB.2.1) (Sigma–Aldrich), 6 His (BD Biosciences), p53, p53 phosphorylated at Ser15 (Cell Signaling) were purchased. Protein knockdown of p53 by RNA interference using siRNA. p53 pub siRNA and Control (non-silencing) siRNA were purchased from Qiagen. Cotransfection of expression vector (pCLIG-RFT, pCLIG) and siRNA (p53 pub. siRNA, scramble siRNA) for U87MG cells (1 104 cells/ml) was performed with Lipofectamine 2000 in 6-well plates for Western blot. Per well, 4 ml lipofectamine 2000 diluted in 100 ml Opti-MEM was applied to a premix consisting of siRNA (20 mM, 4 ml)/Opti-MEM (100 ml), and incubated for 30 min. The whole mixture was added to the medium, which then was changed to DMEM 3 h after transfection. Cells were incubated for 24–48 h before analysis of knockdown p53 by Western blot as described above. For immunocytochemistry, cotransfection of expression vector (pCLIG-RFT, pCLIG) and siRNA to U87MG cells (5 105 cells/ml) were performed with Lipofectamine 2000 in 8-well culture slides. Briefly, for 1 105 cells, 2 ml Lipofectamine 2000 diluted in 30 ml OptiMEM was applied to a pCLIG-RFT or pCLIG (1 lg)/siRNA (20 lM, 2 ll)/Opti-MEM (30 ll) mixture, and incubated for 30 min. The entire mixture was added to the cell-containing medium, which then was changed to DMEM 3 h after transfection. Cells were incubated for 24 h before immunocytochemistry were performed as described above and counting of His positive cells.
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Results
RFT induces G1–S arrest preceding apoptosis
Overexpression of RFT induces apoptosis in glioma cell
We next tested whether overexpression of RFT influence cell cycle. Cell cycle was examined by fluorescence activated cell sorting (FACS) analysis. The FACS analysis demonstrated that overexpression of RFT blocked G1–S progression in U87MG (Fig. 3A) but not in U251MG (data not shown). To determine the effect of G1–S progression, we performed bromodeoxyuridine (BrdU) incorporation assay in 24–36 h after the retrovirus infection. Overexpression of RFT disturbed BrdUincorporation in U87MG (Fig. 2B) and U138MG, but neither U251, T98G nor HeLa cell (data not shown). Cell death was examined by TUNEL assay. TUNEL positive cells were increasing in RFT-expressed U87MG and U138MG in time-dependent manner after retrovirus infection (Fig. 4). However, TUNEL positive cells did neither increase in U251MG nor T98G after overexpression of RFT (data not shown). These results suggested that RFT could induce apoptosis following G1–S arrest in glioma cell.
To test whether RFT induces apoptosis in various glioma cell lines, we overexpressed RFT in glioma cell lines, U87MG, U138MG, U251MG, and T98G, using a replication-incompetent retrovirus CLIG system [11]. Expressions of His-tagged RFT and GFP were simultaneously confirmed 24 h after retrovirus infection to all glioma cell lines. Immunofluorescence study demonstrated that RFT was localized in the nucleus of all cell lines (Fig. 1). In cell growth assay using MTS, overexpression of RFT-induced cell death in U87MG and U138MG 48 h after retrovirus infection but neither in U251MG, T98G (Fig. 2A) nor HeLa cell (data not shown), although the infection efficiencies were the same in all cell lines. Previous report demonstrated that the reduction of FGF-2 caused apoptosis of RFTexpressed U87MG. Therefore, we evaluated whether FGF-2 took a part of apoptosis induced by RFT. However, the addition of FGF-2 by various concentrations did not rescue apoptosis in RFT-expressed U87MG nor U138MG (Fig. 2A). We investigated whether there were any differences of RFT expression in each glioma cell lines. We found that RFT protein expressed in U138MG and T98G but neither in U87MG nor U251MG by RT-PCR using the specific exon of RFT (Fig. 2B). These results indicated that RFT-induced apoptosis was not artificial phenomenon by its overexpression, and that it was not directly caused by the FGF-2 reduction.
RFT phosphorylates p53 and increases expression of p21Waf1 We tried to find the mechanism of RFT regulation in G1–S arrest and apoptosis. The p53 protein is ubiquitously involved in G1–S arrest and/or apoptosis of the most cells, mainly through the control of p21Waf1 expression level [14,17,20]. It has been shown that U251MG and T98G have mutated non-functioningtype p53, but U87MG and U138MG have functioning-
Fig. 1. Localization of overexpressed RFT in glioma cell lines. Simultaneous expressions of RFT and GFP were confirmed 24 h after virus infection to glioma cell lines. By the detection of His-tagged RFT, the localization of RFT was in the nucleus of all cell lines. Scale bars, 20 lm.
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Fig. 2. Effect of cell growth by RFT and its differential expression level in each glioma cell lines. (A) Overexpression of RFT by retrovirus CLIG-HisRFT inhibited cell growth and induced cell death in U87MG and U138MG, but neither U251MG nor T98G 48 h after infection. The addition of FGF-2 (1 lg/ml) did not rescue the obstacle of cell growth in RFT expressed U87MG nor U138MG. % control; RFT overexpressed cells/control cells X100 (B) RFT highly expressed in some glioma cell lines, U138MG and T98G, but neither U87MG nor U251MG.
Fig. 3. G1–S arrest and apoptosis induced by overexpression of RFT. (A) FACS analysis demonstrates that RFT prevented G1–S progression in U87MG 48 h after the infection. (B) RFT downregulated BrdU incorporations in U87MG cell but not U251. ***p < 0:001; scale bars, 50 lm.
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transient expression and knockdown method that U87MG cell was introduced the expression vector CLIG-RFT and small interfering RNA (siRNA) of p53 by using lipofection method, previously described method [2]. The experiment revealed that RNAi knockdown of p53 abolished RFT-induced apoptosis in U87MG cell (Fig. 6A). Moreover, RNAi knockdown of p53 inhibited the increasing expression of p21Waf1 (Fig. 6B). Our findings indicated that RFT regulates G1–S transition and apoptosis via the activation of p53/ p21Waf1 pathway, although it was still unclear how RFT activated p53/p21Waf1 pathway.
Discussion
Fig. 4. Detection of apoptosis induced by RFT. TUNEL staining indicate that TUNEL positive cells were increasing in RFT-expressed U87MG and U138MG in a time-dependent manner but control virus did not, whereas only a few TUNEL positive cells were seen at 24 h in both infected cells. Scale bars, 50 lm.
type p53. The p53 reporter activities were reported to be 0, 0, 36, and 9 in U251MG, TG98, U87MG, and U138MG, respectively [12,23,29]. Therefore, we tested the expression and phosphorylation levels of p53 in the RFT regulation of G1–S arrest and apoptosis in U87MG with wild-type p53. We found that the expression levels of p53 were increased and its phosphorylation at Ser-15 residue was highly increased in RFT-expressed U87MG. Moreover, the expression level of p21Waf1 was also increased (Fig. 5). We evaluated whether p53 pathway was essential for RFT regulation of cell cycle and apoptosis. We used the
Fig. 5. Activation of p53 p21Waf1 pathway by RFT. Overexpression of RFT upregulated the expression level and the phosphorylation of p53 at Ser-15 residue. Moreover, RFT increased the expression level of p21Waf1 in U87MG.
In the previous report, RFT decreased FGF-2 promotor activity in vitro and it was identified as a repressor of FGF-2 transcription. Overexpression of RFT using adeno-associated virus led glioma cell to FGF-2 reduction and apoptosis [28]. It has been widely accepted that FGF-2 signaling is important for survival of most types of cell, expressing both the FGF-2 and the FGF-2 receptor genes [1,9]. A neutralizing antibody against basic FGF-2 or blocking peptides of FGF-2 receptor has been reported to inhibit the growth of U87MG, U251MG, T98G, and HeLa cells [15,19,27]. However, we found that RFT-induced apoptosis was not rescued by the addition of FGF-2 in U87MG and U138MG, and that overexpression of RFT did not induce apoptosis in U251MG, T98G nor HeLa cell. High expression of endogenous full-length RFT was identified in U138MG and T98G. These indicated that RFT could not always suppress the expression of FGF-2 in vivo and that unknown mechanisms might activate RFT to repress the FGF-2 transcription. The tumor suppressor protein p53 is known as ubiquitous regulator of cell cycle and/or apoptosis. In glioma cell lines, U251MG and T98G have mutatedtype p53, which is known to be loss of function, but U87MG and U138MG has functioning-type p53 (wildtype p53 in U87MG, mutated type in U138MG) [12,29]. In this study, RFT regulated cell cycle through the activation of p53 by phosphorylation at ser-15 and the upregulation of p21Waf1 expression level, and the knockdown of p53 prevented apoptosis in RFT-overexpressed U87MG. The p53 is essential for the regulation of cell cycle and apoptosis by RFT. Therefore, overexpression of exogenous RFT did not induce apoptosis in U251MG nor T98G with non-functioningtype p53. It is still unclear how RFT activate p53 pathway. RFT might indirectly activate p53 by a transcription of some genes, which are involved in activation of p53, or it could activate directly. Recent studies has shown that members of the phosphoinositide-3 kinase-
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Fig. 6. RNAi knockdown of p53 abolished RFT-induced apoptosis in U87MG (A,B). By the transient expression and knockdown method, U87MG was introduced the both of CLIG-RFT and siRNA-p53. RNAi knockdown of p53 abolished RFT-induced apoptosis in U87MG 48 h after the transfection. (C) RNAi knockdown of p53 inhibited the expression of p21Waf1 as well as that of p53. ***p < 0:001; **p < 0:025; scale bars, 50 lm.
related kinase (PIK) superfamily, including the TOR subfamily, the ataxia telangiectasia mutated (ATM) subfamily, and DNA-PK, regulate cell-cycle progression and DNA repair in eukaryotic cells. ATM and ATR participate in the activation of cell cycle checkpoints induced by DNA damage [13,30]. ATM has been shown to be involved in the DNA damage-induced phosphorylation of p53 at Ser-15 [3,5]. Ser-15 is a functionally important residue within the p53 amino-terminal region [7], and phosphorylation of Ser-15 represents an early cellular response to a variety of genotoxic stresses [24,26]. The p53 phosphorylated at Ser-15 displayed reduced binding to the inhibitor protein MDM2 [25]. Because the association with MDM2 targets p53 for proteosome-mediated degradation and inhibits its transactivating function [10,16], Ser-15 phosphorylation promotes both the accumulation and functional activation of p53 in response to DNA damage. Two RFT spliced variants with deletions in the putative DNA-binding domain failed to induce G1–S arrest and apoptosis. RFT need to bind DNA with DNA-binding domain when it function. Therefore, there might be a link between RFT and PIK family involving the sense of DNA damage and the regulation of G1–S transition. In conclusion, RFT regulates cell cycle and apoptosis in p53-dependent manner. For these regulations, RFT phosphorylates p53 at Ser-15 and upregulates the expression level of p21Waf1 . Although its mechanism is not still unknown, the specific exon of RFT might act on the activation of p53 pathway. Our results are suggested that RFT could not only function for the FGF-2 repressor but also the maintenance of genome stability through p53/p21Waf1 pathway.
Acknowledgments The authors thank Furuyashiki T, Thumkeo D, and Yamana N (Dept. of Pharmacology, Kyoto University) for discussion, Dr. Kafri T (Univ. North Carolina) for pcDNA-VSV-G. This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (N.H., K.N., J.T., and R.K.) and the Ministry of Health, Labour and Welfare (N.H.). Y.A. is a postdoctoral fellow from the Japan Society for Promotion of Science.
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