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SDI1 is upregulated due to increased mRNA stability during hydroxyurea-induced senescence of human fibroblasts
Mechanisms of Ageing and Development 126 (2005) 1255–1261 www.elsevier.com/locate/mechagedev
p21WAF/CIP1/SDI1 is upregulated due to increased mRNA stability during hydroxyurea-induced senescence of human fibroblasts Hyun-Seok Kim a,1, Eui-ju Yeo b,1, Seong-Hoon Park a, Joo-In Park c, Sang-Chul Park d, Jong-Yeon Shin a, Min-Ju Kim a, Soo-Jin Oh a, Moo-Ho Won f, Tae-Chun Kang f, Jae-Bong Park a, Jaebong Kim a, Jong-Il Kim a, Hyun-Yong Lee e, Jae-Yong Lee a,* a
Department of Biochemistry, College of Medicine, Hallym University, 1 Okchon-dong, Chuncheon, Gangwon-do 200-702, South Korea b Department of Biochemistry, Gachun University College of Medicine, Ganghwa, South Korea c Department of Biochemistry, Dong-A University College of Medicine, Pusan, South Korea d Department of Biochemistry and Molecular Biology, Seoul National University Medical School, Seoul, South Korea e School of Biotechnology and Bioengineering, Gangwon University, Chuncheon, South Korea f Department of Anatomy, College of Medicine, Hallym University, 1 Okchon-dong, Chuncheon, Gangwon-do 200-702, South Korea Accepted 6 July 2005 Available online 18 August 2005
Abstract Hydoxyurea induces senescence-like growth arrest in normal human fibroblasts. p21WAF/CIP1/SDI1, a cyclin dependent kinase inhibitor, was found to be upregulated during this growth arrest. Levels of p21WAF/CIP1/SDI1 protein and mRNA were increased nine-fold by hydroxyurea in these cells. In order to determine whether p21WAF/CIP1/SDI1 mRNA is increased by hydroxyurea at the transcriptional level, human fibroblast cells were transfected with reporter constructs containing a p21WAF/CIP1/SDI1 promoter fragment and then treated with hydroxyurea. The luciferase activities in the reporter-transfected fibroblast cells were not increased by hydroxyurea, indicating that p21WAF/CIP1/SDI1 transcription was not elevated by hydroxyurea. The half-life of the p21WAF/CIP1/SDI1 mRNA was increased by 2.5-fold but that of p21WAF/CIP1/SDI1 protein was not. Our results suggest that increased mRNA stability is the major mechanism of p21WAF/CIP1/SDI1 elevation in the hydroxyurea-induced growth arrest of human fibroblasts. # 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Hydroxyurea; p53; p21WAF/CIP1/SDI1; mRNA stability; Human fibroblasts; Senescence
1. Introduction Hydroxyurea has been used as an effective anti-tumor agent for malignant solid tumors and is still the principle drug for patients with myeloproliferative syndrome (Navarra and Preziosi, 1999). Hydroxyurea inhibits ribonucleotide reductase by destroying the tyrosine free radical essential for its enzyme activity (Karkoff et al., 1968; Moore and Hurlbert, 1985; Timson, 1975). The inhibition of ribonucleotide reductase results in depletion * Corresponding author. Fax: +82 33 244 8425. E-mail address: [email protected] (J.-Y. Lee). 1 They contributed equally to the work.
of intracellular dNTP and DNA synthesis cessation (Timson, 1975). The resulting situation causes growth arrest of human fibroblast cells in culture. Interestingly, these growth-arrested cells showed an elongated flattened morphology typical of senescent fibroblasts (Yeo et al., 2000). Hydroxyurea-arrested cells also showed to express high SenA-b-gal, which has been reported to be expressed only in senescent cells, indicating that growth arrest by hydroxyurea resembles replicative senescence (Yeo et al., 2000). The expression of p21WAF/CIP1/SDI1, a cyclin dependent kinase inhibitor and a downstream effector of p53, was increased after hydroxyurea treatment, but the mechanism was not investigated (Linke et al., 1996; Zhan et al., 1993). The elevation of p21WAF/CIP1/SDI1 and the
0047-6374/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mad.2005.07.002
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induction of growth arrest by hydroxyurea treatment correlated well, suggesting that the elevation of p21WAF/ CIP1/SDI1 is important for growth arrest. p21WAF/CIP1/SDI1 plays a crucial role in mediating growth arrest in response to a number of signals associated with DNA damage, TGFb signaling, and cell differentiation (Gartel and Tyner, 1998). The expression of the p21WAF/CIP1/SDI1 gene has reported to be regulated at both the transcriptional and post-transcriptional levels (Gartel and Tyner, 1999; Zeng et al., 1997). The transcriptional regulation of p21WAF/CIP1/SDI1 has been extensively investigated and various growth arrest-inducing signals are known to be involved in the transcriptional activation of p21WAF/CIP1/ SDI1 through the p21WAF/CIP1/SDI1 promoter (Gartel and Tyner, 1999), which possesses a number of different regulatory elements that are responsible for different signals (Gartel and Tyner, 1999). Recent evidence suggests that a post-transcriptional mechanism also plays an important role in the regulation of p21WAF/CIP1/SDI1 gene expression, which has been reported to be elevated at the post-transcriptional level by epidermal growth factor and UV light (Johannessen et al., 1999; Esposito et al., 1997; Zeng and El-Deiry, 1996; Li et al., 1996; Schwaller et al., 1995; Gorospe et al., 1998). Although the mechanism determining mRNA stability is poorly understood, it has been reported that the stability of p21WAF/CIP1/SDI1 mRNA is regulated through AU-rich elements (AREs) located in the 30 untranslated region (UTR), and by RNA-binding proteins recognizing these elements (Joseph et al., 1998; Liu et al., 2000; Wang et al., 2000). We attempted to determine how the p21WAF/CIP1/SDI1 level is elevated in human fibroblasts treated with hydroxyurea. Here, we report that the elevated expression of p21WAF/CIP1/SDI1 mRNA and protein in normal human fibroblasts cells treated with hydroxyurea is mediated via enhanced p21WAF/CIP1/SDI1 mRNA stability.
2.2. Plasmid preparations A plasmid containing 2.4 kb of human wild-type p21WAF/CIP1/SDI1Waf1/Cip1 promoter (pWWP-Luc) was provided by Dr. B. Vogelstein (Johns Hopkins University). A 50 deletion mutant containing a 1.2 kb of p21WAF/CIP1/SDI1 promoter was prepared by PCR from pWWP-Luc plasmid. The PCR product was digested with HindIII and BglII and cloned into pGL2-basic vector. To generate a pGL-Dp53 construct with a deletion of only the distal p53-binding site, pGL-2.4 was first prepared by digesting plasmid (pWWPLuc) with HindIII and then by inserting the HindIII fragment into pGL2-basic vector (pGL-2.4). The resulting plasmid was then digested with SacI to remove the distal p53 site and the longer SacI fragment was religated. 2.3. Transient transfection and luciferase assay Primary human embryonic fibroblasts were transfected with p21WAF/CIP1/SDI1 promoter reporter constructs using lipofectamine (Gibco BRL). Briefly, cells were seeded at a density of 4 105 cells into 60 mm culture dishes 24 h prior to transfection. After incubating DNA–lipid complexes with cells for 5 h, cells were washed with serum-free DMEM. Fresh medium was then added and incubation continued for 24 h. Cells were treated with 600 mM of hydroxyurea for 48 h and then lysed in extraction buffer (0.2 M Tris–HCl, pH 8.0, 0.1% Triton X-100). The resulting supernatant obtained by centrifugation was used to measure luciferase activity according to the manufacturer’s instructions (Promega) using a luminometer (Lumat LB 9507, EG and G Berthold, Germany). pCMV-bGAL, a plasmid containing lac Z, was cotransfected with the p21WAF/CIP1/SDI1 promoter-reporter constructs to normalize transfection efficiency. Three independent experiments were performed to obtain the standard deviation. 2.4. Western blot analysis
2. Material and methods 2.1. Cell culture A primary human embryonic fibroblast cell line established from an abortuse at Hallym University Hospital was used for this study. Primary human embryonic fibroblasts were cultured in DMEM containing 10% fetal bovine serum at 37 8C in a humidified atmosphere containing 5% CO2. Cells were detached by incubation in 0.05% EDTA/0.025% trypsin and replated at a split ratio of 1:4 every two or three days. For hydroxyurea (Sigma) treatment, human embryonic fibroblast cells of 16 population doublings (PDs) were seeded at a density of 5 105 cells in 100 mm culture dishes. Hydroxyurea was added to the culture at about 70% confluence and cells were incubated for the appropriate time periods.
Cells were incubated in lysis buffer (50 mM Tris–HCl, pH 7.4, 100 mM NaCl, 5 mM EDTA, pH 8.0, 10% glycerol, 1% NP40) containing a mixture of protease inhibitors (5 mg/ ml leupeptin, 5 mg/ml aprotinin, 1 mM PMSF) and lysed by sonication with three bursts of 30 s on ice. After centrifugation, the supernatant was stored at 70 8C or used for Western blot analysis. Twenty micrograms of each lysate was separated by SDS-PAGE, transferred to an Immobilon-p (polyvinylidine difluoride) membrane filter, and blocked with 5% nonfat-powdered milk in TBST (50 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Tween-20). The membrane filter was then incubated with the appropriate dilution of anti-p21WAF/CIP1/SDI1 antibody (Pharmingen, clone sx118) (Mahyar-Roemer and Roemer, 2001; Radhakrishnan et al., 2004) and anti-actin antibody (mAb JLA20 from Developmental Studies Hybridoma Bank, USA), and visualized using an ECL system (Amersham). Relative band
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intensities were determined by scanning densitometry (Molecular Dynamics), and anti-actin antibody and Coomassie blue R-250 staining were used to verify the equal loading of proteins into the wells of the polyacrylamide gel. 2.5. Northern blot analysis Total RNA was prepared using RNAzol B reagent (Cinna/ TEL-TEST) according to the manufacturer’s instructions. Five to 10 mg of RNA was denatured and fractionated by electrophoresis through a 1% agarose gel containing 5% formaldehyde. After transferring to Hybond N+ membranes (Amersham), the RNAs were hybridized to an RNA probe labeled using DIG Northern Starter kit (Boehringer Mannheim). The RNA probe for p21WAF/CIP1/SDI1 contained the coding region for p21WAF/CIP1/SDI1 and a portion of the 30 untranslated-region. 18S rRNA was used to verify the equal loading of RNA in the wells of the polyacrylamide gel. Hybridization and detection were performed using a DIG Northern Starter Kit (Boehringer Mannheim) according to manufacturer’s instructions. The intensities of bands were measured by scanning densitometry (Molecular Dynamics).
3. Results 3.1. Influence of hydroxyurea on levels of p21WAF/CIP1/ SDI1 protein and mRNA Hydroxyurea induces senescence-like growth arrest in normal human fibroblasts and p21WAF/CIP1/SDI1 was reported to be elevated (Yeo et al., 2000). To understand the mechanism underlying p21WAF/CIP1/SDI1 elevation by hydroxyurea, primary normal human embryonic fibroblasts (16 PDs) were treated with various concentrations of hydroxyurea for different times. p21WAF/CIP1/SDI1 protein levels were determined by Western blot analysis (Fig. 1A) and found to be elevated by up to nine-fold by treatment with 200–1000 mM of hydroxyurea for 48 h. Northern blot analysis showed that the p21WAF/CIP1/SDI1 mRNA level was also elevated by up to nine-fold at 48 h by hydroxyurea (Fig. 1B). This result indicates that p21WAF/CIP1/SDI1 elevation by hydroxyurea is regulated at the mRNA level. 3.2. Influence of hydroxyurea on the promoter activity of p21WAF/CIP1/SDI1 To test whether the upregulation of p21WAF/CIP1/SDI1 mRNA by hydroxyurea is regulated at the transcriptional level, reporter constructs containing the p21WAF/CIP1/SDI1 promoter fragments were prepared (Fig. 2A). Reporter constructs were then transiently transfected into human fibroblasts, and luciferase activity was measured. The luciferase activity of the full-length promoter (pGL-2.4) was not stimulated considerably by hydroxyurea (Fig. 2Ba) and pGL-Dp53 containing a deletion of the first p53-binding
Fig. 1. Expression of the p21WAF/CIP1/SDI1 mRNA and protein in human embryonic fibroblasts (16 PD) treated with hydroxyurea. Total RNA and protein were isolated from human embryonic fibroblast cells treated with hydroxyurea and subject to Northern blot analysis for RNA and Western blot analysis for protein. (A) Influence of concentration of hydroxyurea. Cells were treated with different concentrations of hydroxyurea for 48 h. (B) Influence of treated time. Cells were treated with 600 mM of hydroxyurea for various time periods. 18S rRNA and anti-beta-actin antibody were used to verify the equal loading of RNA and protein.
sites and pGL-1.2 lacking both p53-binding sites produced similar results (Fig. 2Bb and c). As a positive control, the full-length promoter (pGL-2.4)-reporter construct was transiently transfected into human fibroblasts cells, and cells were treated with sodium butyrate (5 mM) for 48 h, since sodium butyrate is well known to increase p21 transcription (Sowa et al., 1999; Zeng and El-Deiry, 1996; Xiao et al., 1997). Luciferase activity was increased by sodium butyrate as expected (Fig. 2Ba). These findings indicate that the elevation of p21WAF/CIP1/SDI1 mRNA by hydroxyurea is not regulated at the transcriptional level and that p53 does not affect p21WAF/CIP1/SDI1 transcription, suggesting that p21WAF/CIP1/SDI1 mRNA elevation by hydroxyurea is possibly regulated by a post-transcriptional or translational mechanism. 3.3. Influence of hydroxyurea on the stability of p21WAF/CIP1/SDI1 mRNA and protein In order to determine whether p21WAF/CIP1/SDI1 elevation by hydroxyurea is correlated with the enhanced stability of p21WAF/CIP1/SDI1 mRNA or protein, the stabilities of p21WAF/CIP1/SDI1 mRNA and protein were examined by Northern and Western blot analyses, respectively, using bactin as a control. To examine the stability of p21WAF/CIP1/SDI1 mRNA, human embryonic fibroblasts were treated with
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Fig. 2. Influence of hydroxyurea on transcriptional activity of the p21WAF/CIP1/SDI1 promoter. Human fibroblasts were transfected with a luciferase reporter containing the p21WAF/CIP1/SDI1 promoter. At 5 h after transfection, cells were incubated in fresh medium for 24 h, and then treated with 600 mM hydroxyurea for 48 h. Cell lysate was prepared and used for luciferase assay. (A) Schematic representation of reporter constructs. pGL-2.4 contains 2.4 kb of full-length p21WAF/CIP1/SDI1 promoter. pGL-Ap53 contains mutations in the distal p53-binding site. pGL-1.2 includes only 1.2 kb of p21WAF/CIP1/SDI1 promoter lacking both p53-binding sites. (B) Influence of hydroxyurea on luciferase activity. The relative luciferase activities were analyzed from lysates of human fibroblasts treated with hydroxyurea using luminometer as described in Section 2. As a positive control, pGL-2.4-luciferase construct was transiently transfected into human fibroblasts and sodium butyrate (5 mM) was treated for 48 h.
600 mM of hydroxyurea for 48 h, and then treated with actinomycin D (10 mg/ml) for the following 10 h to prevent transcription. The concentration of actinomycin D treated was optimized in the cultures. Northern blot analysis showed that the stability of p21WAF/CIP1/SDI1 mRNAwas increased 2.5-fold by hydroxyurea, whereas the stability of actin mRNA was unchanged (Fig. 3). To examine the effects of hydroxyurea on the stability of p21WAF/CIP1/SDI1 protein, human fibroblasts were first treated with 600 mM of hydroxyurea for 48 h and then cycloheximide (20 mg/ml) was added for 10 h to prevent the translation. The concentration of cycloheximide was also optimized in the cultures. Western blot analysis revealed that p21WAF/CIP1/SDI1 protein stability was not elevated by hydroxyurea nor was the stability of actin protein (Fig. 4). Since the half-life of mRNA or protein represents the rate of disappearance, a 2.5-fold increase in the half-life of p21WAF/CIP1/SDI1 mRNA seems to be sufficient to produce a 9-fold increase in p21WAF/CIP1/SDI1 mRNA and protein. Therefore, our results indicate that p21WAF/CIP1/SDI1 mRNA is stabilized by hydroxyurea, thus elevating p21WAF/CIP1/SDI1 mRNA and protein levels.
4. Discussion Normal human embryonic fibroblast cells undergo growth arrest with senescence-like morphology when treated with hydroxyurea. Staining of SenA-b-gal, an in vitro and in vivo aging marker, was increased as seen in senescent fibroblast (data not shown). The level of p21WAF/ CIP1/SDI1 was also elevated by treatment with hydroxyurea indicating that hydroxyurea-induced growth arrest is similar to replicative senescence. However, the level of p16INK4a is not elevated differently from replicative senescence. It has not been clear how the level of p21WAF/CIP1/SDI1 is increased by hydroxyurea. Hydroxyurea inhibits the activity of ribonucleotide reductase required for DNA replication and repair, and thereby depletes intracellular dNTP pools. This depletion has been considered to induce the cell cycle arrest (Timson, 1975). Since the depletion of intracellular dNTP pools is a cellular stress, activation of p53, a most important stress activated protein, seems to be involved. This is very likely since it has been reported that depletion of ribonucleotides
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Fig. 3. Influence of hydroxyurea on stability of the p21WAF/CIP1/SDI1 mRNA. Human fibroblasts (16 PD) were incubated in fresh medium containing hydroxyurea (600 mM) for 48 h. Cells were then treated with actinomycin D (10 mg/ml) for various time periods. Total RNA was isolated and p21WAF/CIP1/SDI1 and b-actin mRNA were analyzed at the times indicated by Northern blot analysis as described in Section 2. b-Actin was used as a control to ensure the analysis of mRNA stability using actinomycin D.18S rRNA was used to verify the equal loading of RNA. Intensity of bands in upper panel was measured using densitometer and plotted as percent remaining density in a log scale. t1/2 indicates the half-life that was calculated from the slope of the plot. (A) Stability of the p21WAF/CIP1/SDI1 mRNA and a log scale plot. (B) Stability of the b-actin mRNA and a log scale plot. The blots prepared from untreated extracts were exposed to film longer when compared to the blots prepared from HU-treated extracts to obtain similar band intensity.
Fig. 4. Influence of hydroxyurea on stability of the p21WAF/CIP1/SDI1 protein. Human fibroblasts (16 PD) were incubated in fresh medium containing hydroxyurea (600 mM) for 48 h. Cells were then treated with cycloheximide (20 mg/ml) for various time periods. b-Actin was used as a control to ensure the analysis of protein stability using cycloheximide. The p21WAF/CIP1/SDI1 and b-actin protein were examined by Western blot analysis as described in Section 2. Intensity of bands in upper panel was measured using densitometer and plotted as percent remaining density in log scale. t1/2 indicates the half-life that was calculated from the slope of the plot. (A) Stability of the p21WAF/CIP1/SDI1 protein and a log scale plot. (B) Stability of the b-actin protein and a log scale plot. The blots prepared from untreated extracts were exposed to film longer when compared to the blots prepared from HU-treated extracts to obtain similar band intensity.
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involves the activation of p53 (Linke et al., 1996). p53 is known to activate the transcription of p21WAF/CIP1/SDI1 by binding to p53-binding sites in the p21WAF/CIP1/SDI1 promoter. We tested the transcription of p21WAF/CIP1/SDI1 by luciferase reporter assay. Interestingly, the transcription of p21WAF/CIP1/SDI1 was not activated by hydroxyurea even though the p21WAF/CIP1/SDI1 mRNA and the p21WAF/CIP1/SDI1 protein were increased up to nine-fold. Instead, p21WAF/CIP1/SDI1 turned out to be regulated mostly at the level of mRNA stability. Transient transfection and reporter assay represents the transcription from the exogenously imported DNA. It may not be same as the transcription from the endogenous chromosomal DNA. Since the half-life represents the rate, it appears that 2.5-fold increase in mRNA stability alone is enough to produce 9-fold increase in steady-state RNA level. Similar observations were reported when cells were treated with UV (Gorospe et al., 1998) and retinoid (Li et al., 1996). The transcription of p21WAF/CIP1/SDI1 was not increased when the mouse embryonal fibroblasts were treated with UV (Gorospe et al., 1998). The levels of mRNA and protein were elevated by exposure to UV 25–30-fold. Only the half-life of p21WAF/CIP1/SDI1 mRNA was elevated 2.5–4-fold. Even though they did not measure the stability of protein, they proposed a similar conclusion, Elevation of p21WAF/CIP1/SDI1 by hydroxyurea does not depend on p53. The HeLa cell, a cervical carcinoma cell line that has the integration of human papilloma virus and thereby contains inactive p53 and RB, showed similar elevation of p21WAF/CIP1/SDI1 by hydroxyurea. The result indicates that p21WAF/CIP1/SDI1 can be elevated by hydroxyurea through p53-independent mechanism (data not shown). Our results showed that the treatment of hydroxyurea causes the depletion of intracellular dNTPs, p21WAF/CIP1/SDI1 elevation, and growth arrest. However, it does not involve the activity of p53. Interestingly, the depletion of NTPs is known to involve p53-dependent activation of p21WAF/CIP1/SDI1. Therefore, the result indicates that the depletion of dNTP and the depletion of NTP follow a different cellular response pathway, even though the detailed mechanism is not currently available. How is the p21WAF/CIP1/SDI1 mRNA stabilized by hydroxyurea? Currently, we do not have an answer for this. However, the increased stability of p21WAF/CIP1/SDI1 mRNA has also reported to be involved in growth arrestinducing signals like epidermal growth factor (Johannessen et al., 1999), redox state (Esposito et al., 1997) and UV light (Gorospe et al., 1998). Although the mechanisms determining mRNA stability are not completely understood, it has been reported that the stability of the p21WAF/CIP1/SDI1 mRNA is regulated through AU-rich elements (AREs) located in the 30 untranslated region (UTR) (Joseph et al., 1998; Liu et al., 2000; Wang et al., 2000). RNA-binding proteins like HuR that recognize AREs was also suggested to be involved (Wang et al., 2000). In our preliminary experiments, HuR protein was not affected by HU in the cytosol extracts and total extracts of cells (data not shown).
Therefore, we speculate that the enhanced stabilization of p21WAF/CIP1/SDI1 mRNA by HU may be mediated by other unknown factors. Further detailed characterization of enhanced mRNA stability should be performed and will provide better understanding of the mechanism. Acknowledgements This study was supported by a Research Grant from Department of Welfare and Health of Korean Government (Molecular Ageing Research Center) and a grant from Korea Ministry of Science and Technology (Contract #0101029-12(2002325)) and Gangwon Province.
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p21WAF/CIP1/SDI1 is upregulated due to increased mRNA stability during hydroxyurea-induced senescence of human fibroblasts Hyun-Seok Kim a,1, Eui-ju Yeo b,1, Seong-Hoon Park a, Joo-In Park c, Sang-Chul Park d, Jong-Yeon Shin a, Min-Ju Kim a, Soo-Jin Oh a, Moo-Ho Won f, Tae-Chun Kang f, Jae-Bong Park a, Jaebong Kim a, Jong-Il Kim a, Hyun-Yong Lee e, Jae-Yong Lee a,* a
Department of Biochemistry, College of Medicine, Hallym University, 1 Okchon-dong, Chuncheon, Gangwon-do 200-702, South Korea b Department of Biochemistry, Gachun University College of Medicine, Ganghwa, South Korea c Department of Biochemistry, Dong-A University College of Medicine, Pusan, South Korea d Department of Biochemistry and Molecular Biology, Seoul National University Medical School, Seoul, South Korea e School of Biotechnology and Bioengineering, Gangwon University, Chuncheon, South Korea f Department of Anatomy, College of Medicine, Hallym University, 1 Okchon-dong, Chuncheon, Gangwon-do 200-702, South Korea Accepted 6 July 2005 Available online 18 August 2005
Abstract Hydoxyurea induces senescence-like growth arrest in normal human fibroblasts. p21WAF/CIP1/SDI1, a cyclin dependent kinase inhibitor, was found to be upregulated during this growth arrest. Levels of p21WAF/CIP1/SDI1 protein and mRNA were increased nine-fold by hydroxyurea in these cells. In order to determine whether p21WAF/CIP1/SDI1 mRNA is increased by hydroxyurea at the transcriptional level, human fibroblast cells were transfected with reporter constructs containing a p21WAF/CIP1/SDI1 promoter fragment and then treated with hydroxyurea. The luciferase activities in the reporter-transfected fibroblast cells were not increased by hydroxyurea, indicating that p21WAF/CIP1/SDI1 transcription was not elevated by hydroxyurea. The half-life of the p21WAF/CIP1/SDI1 mRNA was increased by 2.5-fold but that of p21WAF/CIP1/SDI1 protein was not. Our results suggest that increased mRNA stability is the major mechanism of p21WAF/CIP1/SDI1 elevation in the hydroxyurea-induced growth arrest of human fibroblasts. # 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Hydroxyurea; p53; p21WAF/CIP1/SDI1; mRNA stability; Human fibroblasts; Senescence
1. Introduction Hydroxyurea has been used as an effective anti-tumor agent for malignant solid tumors and is still the principle drug for patients with myeloproliferative syndrome (Navarra and Preziosi, 1999). Hydroxyurea inhibits ribonucleotide reductase by destroying the tyrosine free radical essential for its enzyme activity (Karkoff et al., 1968; Moore and Hurlbert, 1985; Timson, 1975). The inhibition of ribonucleotide reductase results in depletion * Corresponding author. Fax: +82 33 244 8425. E-mail address: [email protected] (J.-Y. Lee). 1 They contributed equally to the work.
of intracellular dNTP and DNA synthesis cessation (Timson, 1975). The resulting situation causes growth arrest of human fibroblast cells in culture. Interestingly, these growth-arrested cells showed an elongated flattened morphology typical of senescent fibroblasts (Yeo et al., 2000). Hydroxyurea-arrested cells also showed to express high SenA-b-gal, which has been reported to be expressed only in senescent cells, indicating that growth arrest by hydroxyurea resembles replicative senescence (Yeo et al., 2000). The expression of p21WAF/CIP1/SDI1, a cyclin dependent kinase inhibitor and a downstream effector of p53, was increased after hydroxyurea treatment, but the mechanism was not investigated (Linke et al., 1996; Zhan et al., 1993). The elevation of p21WAF/CIP1/SDI1 and the
0047-6374/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mad.2005.07.002
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induction of growth arrest by hydroxyurea treatment correlated well, suggesting that the elevation of p21WAF/ CIP1/SDI1 is important for growth arrest. p21WAF/CIP1/SDI1 plays a crucial role in mediating growth arrest in response to a number of signals associated with DNA damage, TGFb signaling, and cell differentiation (Gartel and Tyner, 1998). The expression of the p21WAF/CIP1/SDI1 gene has reported to be regulated at both the transcriptional and post-transcriptional levels (Gartel and Tyner, 1999; Zeng et al., 1997). The transcriptional regulation of p21WAF/CIP1/SDI1 has been extensively investigated and various growth arrest-inducing signals are known to be involved in the transcriptional activation of p21WAF/CIP1/ SDI1 through the p21WAF/CIP1/SDI1 promoter (Gartel and Tyner, 1999), which possesses a number of different regulatory elements that are responsible for different signals (Gartel and Tyner, 1999). Recent evidence suggests that a post-transcriptional mechanism also plays an important role in the regulation of p21WAF/CIP1/SDI1 gene expression, which has been reported to be elevated at the post-transcriptional level by epidermal growth factor and UV light (Johannessen et al., 1999; Esposito et al., 1997; Zeng and El-Deiry, 1996; Li et al., 1996; Schwaller et al., 1995; Gorospe et al., 1998). Although the mechanism determining mRNA stability is poorly understood, it has been reported that the stability of p21WAF/CIP1/SDI1 mRNA is regulated through AU-rich elements (AREs) located in the 30 untranslated region (UTR), and by RNA-binding proteins recognizing these elements (Joseph et al., 1998; Liu et al., 2000; Wang et al., 2000). We attempted to determine how the p21WAF/CIP1/SDI1 level is elevated in human fibroblasts treated with hydroxyurea. Here, we report that the elevated expression of p21WAF/CIP1/SDI1 mRNA and protein in normal human fibroblasts cells treated with hydroxyurea is mediated via enhanced p21WAF/CIP1/SDI1 mRNA stability.
2.2. Plasmid preparations A plasmid containing 2.4 kb of human wild-type p21WAF/CIP1/SDI1Waf1/Cip1 promoter (pWWP-Luc) was provided by Dr. B. Vogelstein (Johns Hopkins University). A 50 deletion mutant containing a 1.2 kb of p21WAF/CIP1/SDI1 promoter was prepared by PCR from pWWP-Luc plasmid. The PCR product was digested with HindIII and BglII and cloned into pGL2-basic vector. To generate a pGL-Dp53 construct with a deletion of only the distal p53-binding site, pGL-2.4 was first prepared by digesting plasmid (pWWPLuc) with HindIII and then by inserting the HindIII fragment into pGL2-basic vector (pGL-2.4). The resulting plasmid was then digested with SacI to remove the distal p53 site and the longer SacI fragment was religated. 2.3. Transient transfection and luciferase assay Primary human embryonic fibroblasts were transfected with p21WAF/CIP1/SDI1 promoter reporter constructs using lipofectamine (Gibco BRL). Briefly, cells were seeded at a density of 4 105 cells into 60 mm culture dishes 24 h prior to transfection. After incubating DNA–lipid complexes with cells for 5 h, cells were washed with serum-free DMEM. Fresh medium was then added and incubation continued for 24 h. Cells were treated with 600 mM of hydroxyurea for 48 h and then lysed in extraction buffer (0.2 M Tris–HCl, pH 8.0, 0.1% Triton X-100). The resulting supernatant obtained by centrifugation was used to measure luciferase activity according to the manufacturer’s instructions (Promega) using a luminometer (Lumat LB 9507, EG and G Berthold, Germany). pCMV-bGAL, a plasmid containing lac Z, was cotransfected with the p21WAF/CIP1/SDI1 promoter-reporter constructs to normalize transfection efficiency. Three independent experiments were performed to obtain the standard deviation. 2.4. Western blot analysis
2. Material and methods 2.1. Cell culture A primary human embryonic fibroblast cell line established from an abortuse at Hallym University Hospital was used for this study. Primary human embryonic fibroblasts were cultured in DMEM containing 10% fetal bovine serum at 37 8C in a humidified atmosphere containing 5% CO2. Cells were detached by incubation in 0.05% EDTA/0.025% trypsin and replated at a split ratio of 1:4 every two or three days. For hydroxyurea (Sigma) treatment, human embryonic fibroblast cells of 16 population doublings (PDs) were seeded at a density of 5 105 cells in 100 mm culture dishes. Hydroxyurea was added to the culture at about 70% confluence and cells were incubated for the appropriate time periods.
Cells were incubated in lysis buffer (50 mM Tris–HCl, pH 7.4, 100 mM NaCl, 5 mM EDTA, pH 8.0, 10% glycerol, 1% NP40) containing a mixture of protease inhibitors (5 mg/ ml leupeptin, 5 mg/ml aprotinin, 1 mM PMSF) and lysed by sonication with three bursts of 30 s on ice. After centrifugation, the supernatant was stored at 70 8C or used for Western blot analysis. Twenty micrograms of each lysate was separated by SDS-PAGE, transferred to an Immobilon-p (polyvinylidine difluoride) membrane filter, and blocked with 5% nonfat-powdered milk in TBST (50 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Tween-20). The membrane filter was then incubated with the appropriate dilution of anti-p21WAF/CIP1/SDI1 antibody (Pharmingen, clone sx118) (Mahyar-Roemer and Roemer, 2001; Radhakrishnan et al., 2004) and anti-actin antibody (mAb JLA20 from Developmental Studies Hybridoma Bank, USA), and visualized using an ECL system (Amersham). Relative band
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intensities were determined by scanning densitometry (Molecular Dynamics), and anti-actin antibody and Coomassie blue R-250 staining were used to verify the equal loading of proteins into the wells of the polyacrylamide gel. 2.5. Northern blot analysis Total RNA was prepared using RNAzol B reagent (Cinna/ TEL-TEST) according to the manufacturer’s instructions. Five to 10 mg of RNA was denatured and fractionated by electrophoresis through a 1% agarose gel containing 5% formaldehyde. After transferring to Hybond N+ membranes (Amersham), the RNAs were hybridized to an RNA probe labeled using DIG Northern Starter kit (Boehringer Mannheim). The RNA probe for p21WAF/CIP1/SDI1 contained the coding region for p21WAF/CIP1/SDI1 and a portion of the 30 untranslated-region. 18S rRNA was used to verify the equal loading of RNA in the wells of the polyacrylamide gel. Hybridization and detection were performed using a DIG Northern Starter Kit (Boehringer Mannheim) according to manufacturer’s instructions. The intensities of bands were measured by scanning densitometry (Molecular Dynamics).
3. Results 3.1. Influence of hydroxyurea on levels of p21WAF/CIP1/ SDI1 protein and mRNA Hydroxyurea induces senescence-like growth arrest in normal human fibroblasts and p21WAF/CIP1/SDI1 was reported to be elevated (Yeo et al., 2000). To understand the mechanism underlying p21WAF/CIP1/SDI1 elevation by hydroxyurea, primary normal human embryonic fibroblasts (16 PDs) were treated with various concentrations of hydroxyurea for different times. p21WAF/CIP1/SDI1 protein levels were determined by Western blot analysis (Fig. 1A) and found to be elevated by up to nine-fold by treatment with 200–1000 mM of hydroxyurea for 48 h. Northern blot analysis showed that the p21WAF/CIP1/SDI1 mRNA level was also elevated by up to nine-fold at 48 h by hydroxyurea (Fig. 1B). This result indicates that p21WAF/CIP1/SDI1 elevation by hydroxyurea is regulated at the mRNA level. 3.2. Influence of hydroxyurea on the promoter activity of p21WAF/CIP1/SDI1 To test whether the upregulation of p21WAF/CIP1/SDI1 mRNA by hydroxyurea is regulated at the transcriptional level, reporter constructs containing the p21WAF/CIP1/SDI1 promoter fragments were prepared (Fig. 2A). Reporter constructs were then transiently transfected into human fibroblasts, and luciferase activity was measured. The luciferase activity of the full-length promoter (pGL-2.4) was not stimulated considerably by hydroxyurea (Fig. 2Ba) and pGL-Dp53 containing a deletion of the first p53-binding
Fig. 1. Expression of the p21WAF/CIP1/SDI1 mRNA and protein in human embryonic fibroblasts (16 PD) treated with hydroxyurea. Total RNA and protein were isolated from human embryonic fibroblast cells treated with hydroxyurea and subject to Northern blot analysis for RNA and Western blot analysis for protein. (A) Influence of concentration of hydroxyurea. Cells were treated with different concentrations of hydroxyurea for 48 h. (B) Influence of treated time. Cells were treated with 600 mM of hydroxyurea for various time periods. 18S rRNA and anti-beta-actin antibody were used to verify the equal loading of RNA and protein.
sites and pGL-1.2 lacking both p53-binding sites produced similar results (Fig. 2Bb and c). As a positive control, the full-length promoter (pGL-2.4)-reporter construct was transiently transfected into human fibroblasts cells, and cells were treated with sodium butyrate (5 mM) for 48 h, since sodium butyrate is well known to increase p21 transcription (Sowa et al., 1999; Zeng and El-Deiry, 1996; Xiao et al., 1997). Luciferase activity was increased by sodium butyrate as expected (Fig. 2Ba). These findings indicate that the elevation of p21WAF/CIP1/SDI1 mRNA by hydroxyurea is not regulated at the transcriptional level and that p53 does not affect p21WAF/CIP1/SDI1 transcription, suggesting that p21WAF/CIP1/SDI1 mRNA elevation by hydroxyurea is possibly regulated by a post-transcriptional or translational mechanism. 3.3. Influence of hydroxyurea on the stability of p21WAF/CIP1/SDI1 mRNA and protein In order to determine whether p21WAF/CIP1/SDI1 elevation by hydroxyurea is correlated with the enhanced stability of p21WAF/CIP1/SDI1 mRNA or protein, the stabilities of p21WAF/CIP1/SDI1 mRNA and protein were examined by Northern and Western blot analyses, respectively, using bactin as a control. To examine the stability of p21WAF/CIP1/SDI1 mRNA, human embryonic fibroblasts were treated with
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Fig. 2. Influence of hydroxyurea on transcriptional activity of the p21WAF/CIP1/SDI1 promoter. Human fibroblasts were transfected with a luciferase reporter containing the p21WAF/CIP1/SDI1 promoter. At 5 h after transfection, cells were incubated in fresh medium for 24 h, and then treated with 600 mM hydroxyurea for 48 h. Cell lysate was prepared and used for luciferase assay. (A) Schematic representation of reporter constructs. pGL-2.4 contains 2.4 kb of full-length p21WAF/CIP1/SDI1 promoter. pGL-Ap53 contains mutations in the distal p53-binding site. pGL-1.2 includes only 1.2 kb of p21WAF/CIP1/SDI1 promoter lacking both p53-binding sites. (B) Influence of hydroxyurea on luciferase activity. The relative luciferase activities were analyzed from lysates of human fibroblasts treated with hydroxyurea using luminometer as described in Section 2. As a positive control, pGL-2.4-luciferase construct was transiently transfected into human fibroblasts and sodium butyrate (5 mM) was treated for 48 h.
600 mM of hydroxyurea for 48 h, and then treated with actinomycin D (10 mg/ml) for the following 10 h to prevent transcription. The concentration of actinomycin D treated was optimized in the cultures. Northern blot analysis showed that the stability of p21WAF/CIP1/SDI1 mRNAwas increased 2.5-fold by hydroxyurea, whereas the stability of actin mRNA was unchanged (Fig. 3). To examine the effects of hydroxyurea on the stability of p21WAF/CIP1/SDI1 protein, human fibroblasts were first treated with 600 mM of hydroxyurea for 48 h and then cycloheximide (20 mg/ml) was added for 10 h to prevent the translation. The concentration of cycloheximide was also optimized in the cultures. Western blot analysis revealed that p21WAF/CIP1/SDI1 protein stability was not elevated by hydroxyurea nor was the stability of actin protein (Fig. 4). Since the half-life of mRNA or protein represents the rate of disappearance, a 2.5-fold increase in the half-life of p21WAF/CIP1/SDI1 mRNA seems to be sufficient to produce a 9-fold increase in p21WAF/CIP1/SDI1 mRNA and protein. Therefore, our results indicate that p21WAF/CIP1/SDI1 mRNA is stabilized by hydroxyurea, thus elevating p21WAF/CIP1/SDI1 mRNA and protein levels.
4. Discussion Normal human embryonic fibroblast cells undergo growth arrest with senescence-like morphology when treated with hydroxyurea. Staining of SenA-b-gal, an in vitro and in vivo aging marker, was increased as seen in senescent fibroblast (data not shown). The level of p21WAF/ CIP1/SDI1 was also elevated by treatment with hydroxyurea indicating that hydroxyurea-induced growth arrest is similar to replicative senescence. However, the level of p16INK4a is not elevated differently from replicative senescence. It has not been clear how the level of p21WAF/CIP1/SDI1 is increased by hydroxyurea. Hydroxyurea inhibits the activity of ribonucleotide reductase required for DNA replication and repair, and thereby depletes intracellular dNTP pools. This depletion has been considered to induce the cell cycle arrest (Timson, 1975). Since the depletion of intracellular dNTP pools is a cellular stress, activation of p53, a most important stress activated protein, seems to be involved. This is very likely since it has been reported that depletion of ribonucleotides
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Fig. 3. Influence of hydroxyurea on stability of the p21WAF/CIP1/SDI1 mRNA. Human fibroblasts (16 PD) were incubated in fresh medium containing hydroxyurea (600 mM) for 48 h. Cells were then treated with actinomycin D (10 mg/ml) for various time periods. Total RNA was isolated and p21WAF/CIP1/SDI1 and b-actin mRNA were analyzed at the times indicated by Northern blot analysis as described in Section 2. b-Actin was used as a control to ensure the analysis of mRNA stability using actinomycin D.18S rRNA was used to verify the equal loading of RNA. Intensity of bands in upper panel was measured using densitometer and plotted as percent remaining density in a log scale. t1/2 indicates the half-life that was calculated from the slope of the plot. (A) Stability of the p21WAF/CIP1/SDI1 mRNA and a log scale plot. (B) Stability of the b-actin mRNA and a log scale plot. The blots prepared from untreated extracts were exposed to film longer when compared to the blots prepared from HU-treated extracts to obtain similar band intensity.
Fig. 4. Influence of hydroxyurea on stability of the p21WAF/CIP1/SDI1 protein. Human fibroblasts (16 PD) were incubated in fresh medium containing hydroxyurea (600 mM) for 48 h. Cells were then treated with cycloheximide (20 mg/ml) for various time periods. b-Actin was used as a control to ensure the analysis of protein stability using cycloheximide. The p21WAF/CIP1/SDI1 and b-actin protein were examined by Western blot analysis as described in Section 2. Intensity of bands in upper panel was measured using densitometer and plotted as percent remaining density in log scale. t1/2 indicates the half-life that was calculated from the slope of the plot. (A) Stability of the p21WAF/CIP1/SDI1 protein and a log scale plot. (B) Stability of the b-actin protein and a log scale plot. The blots prepared from untreated extracts were exposed to film longer when compared to the blots prepared from HU-treated extracts to obtain similar band intensity.
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involves the activation of p53 (Linke et al., 1996). p53 is known to activate the transcription of p21WAF/CIP1/SDI1 by binding to p53-binding sites in the p21WAF/CIP1/SDI1 promoter. We tested the transcription of p21WAF/CIP1/SDI1 by luciferase reporter assay. Interestingly, the transcription of p21WAF/CIP1/SDI1 was not activated by hydroxyurea even though the p21WAF/CIP1/SDI1 mRNA and the p21WAF/CIP1/SDI1 protein were increased up to nine-fold. Instead, p21WAF/CIP1/SDI1 turned out to be regulated mostly at the level of mRNA stability. Transient transfection and reporter assay represents the transcription from the exogenously imported DNA. It may not be same as the transcription from the endogenous chromosomal DNA. Since the half-life represents the rate, it appears that 2.5-fold increase in mRNA stability alone is enough to produce 9-fold increase in steady-state RNA level. Similar observations were reported when cells were treated with UV (Gorospe et al., 1998) and retinoid (Li et al., 1996). The transcription of p21WAF/CIP1/SDI1 was not increased when the mouse embryonal fibroblasts were treated with UV (Gorospe et al., 1998). The levels of mRNA and protein were elevated by exposure to UV 25–30-fold. Only the half-life of p21WAF/CIP1/SDI1 mRNA was elevated 2.5–4-fold. Even though they did not measure the stability of protein, they proposed a similar conclusion, Elevation of p21WAF/CIP1/SDI1 by hydroxyurea does not depend on p53. The HeLa cell, a cervical carcinoma cell line that has the integration of human papilloma virus and thereby contains inactive p53 and RB, showed similar elevation of p21WAF/CIP1/SDI1 by hydroxyurea. The result indicates that p21WAF/CIP1/SDI1 can be elevated by hydroxyurea through p53-independent mechanism (data not shown). Our results showed that the treatment of hydroxyurea causes the depletion of intracellular dNTPs, p21WAF/CIP1/SDI1 elevation, and growth arrest. However, it does not involve the activity of p53. Interestingly, the depletion of NTPs is known to involve p53-dependent activation of p21WAF/CIP1/SDI1. Therefore, the result indicates that the depletion of dNTP and the depletion of NTP follow a different cellular response pathway, even though the detailed mechanism is not currently available. How is the p21WAF/CIP1/SDI1 mRNA stabilized by hydroxyurea? Currently, we do not have an answer for this. However, the increased stability of p21WAF/CIP1/SDI1 mRNA has also reported to be involved in growth arrestinducing signals like epidermal growth factor (Johannessen et al., 1999), redox state (Esposito et al., 1997) and UV light (Gorospe et al., 1998). Although the mechanisms determining mRNA stability are not completely understood, it has been reported that the stability of the p21WAF/CIP1/SDI1 mRNA is regulated through AU-rich elements (AREs) located in the 30 untranslated region (UTR) (Joseph et al., 1998; Liu et al., 2000; Wang et al., 2000). RNA-binding proteins like HuR that recognize AREs was also suggested to be involved (Wang et al., 2000). In our preliminary experiments, HuR protein was not affected by HU in the cytosol extracts and total extracts of cells (data not shown).
Therefore, we speculate that the enhanced stabilization of p21WAF/CIP1/SDI1 mRNA by HU may be mediated by other unknown factors. Further detailed characterization of enhanced mRNA stability should be performed and will provide better understanding of the mechanism. Acknowledgements This study was supported by a Research Grant from Department of Welfare and Health of Korean Government (Molecular Ageing Research Center) and a grant from Korea Ministry of Science and Technology (Contract #0101029-12(2002325)) and Gangwon Province.
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