From the Cardiovascular Research Group, School of Animal and Microbial Sciences, The University of Reading, Reading, Berkshire RG6 6AJ, United Kingdom
Received for publication, December 11, 2002
, and in revised form, March 18, 2003.
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ABSTRACT |
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INTRODUCTION |
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Previously, we have reported that a partial re-activation of specific components of the cell cycle machinery occurs during the development of cardiac hypertrophy, leading to transition of myocytes through the G1/S cell cycle checkpoint (2, 4). E2F is a family of transcription factors that are known to play a pivotal role in the G1/S phase transition of the cell cycle (5, 6); however, very little is known about the potential role(s) of these transcription factors in cardiac hypertrophy.
Structurally and functionally, the E2F transcription factors can be divided into three main categories as follows: (i) E2Fs 13, which are involved in proliferation; (ii) E2F-4 and E2F-5 that are thought to play a role in differentiation; and (iii) E2F-6, which is described as a transcriptional repressor (7). For full transcriptional activity, the E2F transcription factors heterodimerize with a DP partner protein, of which two mammalian forms have been described, namely DP-1 and DP-2 (8). The transcriptional activity of E2F members is sterically regulated by the pRb family of pocket proteins that consists of pRb, p107, and p130 (5, 7). E2F activation occurs when cyclin D-cyclin-dependent kinase (CDK) 4/6 complexes in the G1 phase of the cell cycle phosphorylate the pRb pocket proteins, which then dissociate from the E2F-DP complex, resulting in E2F-mediated gene transcription and progression to the S phase of the cell cycle (5, 6). Indeed, various studies have shown that E2F is instrumental in cardiac myocyte cell cycle progression. Thus, Flink et al. (11) showed that a switch occurs from the E2F-p107 complex in proliferating fetal myocytes to E2F-p130 in 2-day-old neonatal cells. Also, von Harsdorf et al. (13) showed that adenoviral delivery of E2F-1 drove a significant number of adult cardiac myocytes into the S phase of the cell cycle; Agah et al. have reported similar findings (12).
Clinically, E2F has proved to be a viable target in the treatment of certain vasculoproliferative diseases. Thus, Mann et al. (10) showed that blocking E2F-1 function in human vein grafts ex vivo, using a decoy oligonucleotide strategy, reduced the incidence of neointimal hyperplasia and subsequent graft failure. The importance of E2F in the heart was shown recently by Cloud et al. (14), who reported that E2F-3a and E2F-3b null mice die from symptoms of congestive heart failure during development, indicating a crucial role for E2F3 in normal cardiac growth. Although much is known about E2F function in a variety of diseases, currently no study has characterized expression and/or inhibited E2F function during the development of cardiac myocyte hypertrophy.
In the present study, we have determined the expressions of E2F and DP family members at the mRNA and protein levels in myocytes obtained from the developing rat heart. In addition, we have determined the expressions and activities of these molecules during the development of myocyte hypertrophy in vitro and shown that blocking E2F function with a peptide that abrogates E2F-DP heterodimerization (15) inhibits the development of hypertrophy. Our results suggest that E2F plays an important role in cardiac myocyte growth and that targeting these transcription factors could provide us with an effective therapy for treating detrimental left ventricular hypertrophy (LVH) leading to heart failure.
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EXPERIMENTAL PROCEDURES |
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Peptide StudiesE2F blocking and control peptides were used at concentrations of 30 µM in myocyte media as described by Bandara et al. (15). Peptide sequences were as follows: AH2, RQIKIWFQNRRMKWKKRRRVYDALNVLMAMNIISK; AHS2, RQIKIWFQNRRMKWKKDRVKAVRNLMIASYRNMLI; and ANT, RQIKIWFQNRRMKWKK.
Flow Cytometric AnalysisTwo-color flow cytometric analysis was performed as described by Poolman and Brooks (1), except that BrdUrd was added directly to myocyte media at a final concentration of 10 µM. Also, pepsin was omitted when myocytes were exposed to 0.1 M HCl and cells were incubated for 10 min. Finally, the staining solution was modified by replacing Isoton with phosphate buffered saline.
Isolation of Total RNA, Semi-quantitative, and Quantitative RT-PCR AnalysesFetal, neonatal, and adult myocytes were homogenized in TRI Reagent (1 ml per 50100 mg of tissue). Total RNA was then prepared from the sample in accordance with the manufacturer's instructions (Sigma). RT-PCR was performed on 5 µg of DNase-treated total RNA using AMV Reverse Transcriptase (Promega) and oligo(dT). PCR analysis was carried out using the following primer sequences: E2F-1, sense 5'-TTCTTGGAGCTGCTGAGCC-3', antisense 5'-TGGTGATGTCATAGATGCG-3'; E2F-2, sense 5'-ATCCAGTGGGTAGGCAGG-3', antisense 5'-TGGGCACAGGTAGACTTC-3'; E2F-3, sense 5'-TGGACCTCAAACTGTTAACCG-3', antisense 5'-GGAGGCAGTAAGTTCACAA-3'; E2F-4, sense 5'-TGCTTGACCTCAAGCTGG-3', antisense 5'-CTCCAGCAAAGCATCTGC-3'; E2F-5, sense 5'-ACCACCAAGTTCGTGTCGTTGC-3', antisense 5'-GTTATGATGAACTGAAGCCTGC-3'; DP-1, sense 5'-GGATCTGGTAACATGGCA-3', antisense 5'-GGATATGATGTTCATGGC-3'; DP-2, sense 5'-TGCAGCATCTCCAGTGAC-3', antisense 5'-CAGAGAGCATTTGCCTGAC-3'; GAPDH, sense 5'-CCTTCATTGACCTCAAC-3', antisense 5'-AGTTGTCATGGATGACC-3'; ANF, sense 5'-ATGGGCTCCTTCTCCATCAC-3', antisense 5'-TCTTCGGTACCGGGAAGCT-3'; BNP, sense 5'-AGACAAGAGAGAGCAGGACACC-3', antisense 5'-CTTGAACTATGTGCCATCTTGG-3'. PCR products were separated on 1% (w/v) agarose/TAE gels. Bands were stained with ethidium bromide and visualized under ultraviolet light. Real-time quantitative PCR analysis was performed using ABsoluteTM QPCR ROX mix (ABgene) and a GeneAmp® 5700 sequence detector (Applied Biosystems). Rat ANF was amplified using sense (5'-CGGACTAGGCTGCAACAGCT-3') and antisense (5'-CCAGGAGGGTATTCACCACCT-3') oligonucleotides and detected using an ANF probe (5'-FAM-CGGTACCGAAGATAACAGCCAAATCTGCT-TAMRA-3'). Real-time PCR analysis was normalized using the rodent GAPDH control reagent kit (Applied Biosystems) as recommended by the manufacturer.
Immunoblot AnalysisProtein was prepared from freshly isolated fetal, neonatal, and adult rat ventricular myocytes as described by Brooks and co-workers (16) except that 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF; 0.3 mM), leupeptin (10 µg/ml), and aprotinin (2 µg/ml) were used as protease inhibitors. Following separation in 12% SDS-polyacrylamide gels, proteins were transferred to nitrocellulose membranes (Amersham Biosciences) before incubation with primary antibodies for 2 h at room temperature. Antibodies (Santa Cruz Biotechnology) directed against either E2F-1 (C-20), E2F-2 (C-20), E2F-3a (N-20), E2F-4 (C-20), E2F-5 (MH-5), DP-1 (K-20), or DP-2 (C-20 and N-20) were used at a dilution of 1:1000 in Tris-buffered saline (TBS)/0.1% Tween 20 containing 1% milk powder. Membranes were then incubated in goat anti-rabbit- or goat anti-mouse-horseradish peroxidase-conjugated antibody (1:4000) for 1 h. Finally, all washes were carried out in TBS/0.1% Tween 20 for 10 min, except the final wash, which was TBS alone.
Luciferase AssaysNeonatal myocytes were plated in 6-well tissue culture dishes at 2.5 x 105/well and left overnight in 5% FCS myocyte medium containing 500 µM BrdUrd. Cells then were co-transfected overnight with either 0.2 µg of a cyclin E promoter-firefly luciferase construct or control vector (lacking the cyclin E promoter) and 0.01 µg of vector encoding the Renilla luciferase enzyme (Promega) using Effectene® transfection reagent (Qiagen). Cells were serum-starved for 24 h and then treated with various hypertrophic agonists for 48 h, after which transfected cells were collected and assayed using a Dual-LuciferaseTM assay kit (Promega).
Electromobility Shift Assay (EMSAs)EMSAs were performed according to La Thangue et al. (17), except that poly(dI-dC) was used as the nonspecific competitor. The E2F binding sequence used was 5'-TAGTTTTCGCGCTTAAATTTGA-3'. Myocyte nuclear extracts (20 µg) were used in each assay, and appropriate competition studies were performed.
Statistical AnalysisAutoradiographs and photographs were scanned and analyzed using the Quantity One® densitometric computer analysis program (Bio-Rad). Data was subjected to one-way analysis of variance (ANOVA), and statistical significance was assessed using the Bonferroni t test. p values of <0.05 were considered to be significant.
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RESULTS |
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Expression of E2F and DP Family Members during the Development of Myocyte HypertrophyHaving established that E2F and DP family members are expressed differentially during normal cardiac development, we next determined whether the regulation of these molecules was similarly altered during the development of myocyte hypertrophy. Using an in vitro model of hypertrophy, cardiac myocytes were stimulated for 24 h with one of two different hypertrophic stimuli, 20% FCS or 100 µM phenylephrine (PE). To confirm that hypertrophy had been induced, we measured the induction of the hypertrophic marker, ANF, by real time PCR, cell cycle progression by flow cytometry and cell size (Fig. 2). Fig. 2a shows that ANF was induced significantly in cells treated for 24 h with FCS (4-fold) and PE (7-fold) compared with the levels expressed in control cells maintained in 0% FCS. Flow cytometric analysis was performed on control and treated cell populations using the propidium iodide/BrdUrd double staining method to accurately determine the cell cycle profiles of these cells. Fig. 2b shows that over 80% of the cells maintained in 0% FCS were in G1 with 5% in the S phase. However, upon stimulation with FCS for 24 h, the S phase population of cells increased to 20%, with a proportional decrease of cells in the G1 phase. PE stimulation also increased the S phase population by 10%, with a proportional decrease of cells in the G1 phase. Thus, a significant number of cells treated with different hypertrophic agonists progress from G1 into S phase, consistent with a partial re-activation of the cell cycle during the development of hypertrophy (4). Finally, Fig. 2c shows that cell size was increased significantly in cultures treated with either 20% FCS or 100 µM PE, compared with control cells (0% FCS). Taken together, these analyses confirm that a significant level of hypertrophy was induced in cardiac myocytes stimulated with our different stimuli.
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We next determined the expression patterns of E2F and DP family members during the development of myocyte hypertrophy. Concentrating on protein expression of E2F and DP family members, Fig. 3 shows that the E2F-1 protein was up-regulated following stimulation with 20% FCS for 24 h, whereas 100 µM PE did not regulate E2F-1 protein levels when compared with controls. A similar result was observed 48 h following agonist stimulation (data not shown). E2F-3a and -4 and DP-1 proteins were all up-regulated following stimulation with FCS or PE. Protein expression of DP-2 in stimulated and non-stimulated cells was very low (determined using both N- and C-terminus-directed DP-2 antibodies). This observation confirms our previous developmental data, which showed that freshly isolated neonatal myocytes contain low amounts of DP-2 (Fig. 1b). Interestingly, E2F-5 protein was not regulated in cells stimulated with PE at any time point, whereas levels were down-regulated significantly following 20% FCS stimulation at both 24 (Fig. 3) and 48 h (data not shown) post-stimulation. In accordance with our developmental results, the E2F-2 protein was not detectable in stimulated or non-stimulated cells. This differential pattern of regulation suggests either distinct functional roles for individual E2F family members and/or a temporal pattern of expression during the development of hypertrophy.
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Changes in E2F Activity in Cardiac Myocytes during the Development of HypertrophyBecause our initial studies showed that the E2F and DP proteins are regulated differentially during the development of cardiac myocyte hypertrophy, we next determined whether the transcriptional activities and DNA binding activities of these proteins was similarly altered during this process by using a combination of dual luciferase assays and EMSAs. We measured global E2F activity in myocytes by transfecting a construct containing the firefly luciferase gene under transcriptional control of the mammalian cyclin E promoter into cultured neonatal myocytes. A second construct, containing the Renilla luciferase gene under transcriptional control of the cytomegalovirus (CMV) promoter, was co-transfected into myocytes to provide a baseline luminescence which could be used to normalize E2F activity and provide an overall ratio of E2F-mediated luciferase activity. Fig. 4a shows that E2F activity was doubled when myocytes were stimulated to undergo hypertrophy with FCS, whereas PE increased E2F activity 6-fold compared with levels found in 0% FCS control cells 48 h post-stimulation. We confirmed that global E2F-DNA binding was increased following treatment with hypertrophic agents by performing a series of EMSAs (Fig. 4b) 24 h post-stimulation. The results of these studies confirmed those from the dual luciferase assays and showed an up-regulation in E2F-DNA binding following stimulation of myocytes with 20% FCS or 100 µM PE. To ensure probe specificity in EMSAs, appropriate competition studies were performed with an unlabeled E2F probe and probes designed to detect Sp1 and PPAR-
(data not shown). Two E2F bands were visualized in EMSAs, namely "free E2F" that describes the E2F-DP heterodimer and "bound E2F" that describes E2F-DP in complex with pocket proteins. A nonspecific band (ns in Fig. 4), which was not competed out in appropriate competition studies, was also observed near the unincorporated nucleotide front.
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Blocking E2F Activity Prevents the Induction of HypertrophyBecause E2F activity is increased during the development of cardiac myocyte hypertrophy (Fig. 4), we next determined whether inhibiting E2F activity would affect this process. E2F-DP heterodimerization is essential for transcriptional E2F activity, and its function can be blocked completely by inhibiting this dimerization. Indeed, a specific peptide sequence named "AH2," targeted to the DEF box region of the heterodimerization domain of E2F, has been shown previously to inhibit the growth of tumor cells consistent with its inhibition of E2F activity (15). We have used this same peptide sequence to block E2F function in myocytes stimulated to undergo hypertrophy with 20% FCS or 100 µM PE. Peptides were N-terminally linked to the penetratin sequence to aid delivery into intact cells (19). The penetratin sequence (referred to as "ANT") and a scrambled sequence based upon AH2 (termed AHS2) were included as negative controls for this study because they do not inhibit E2F function. Using a combination of semi-quantitative (Fig. 5a) and quantitative (Fig. 5b) PCR analyses, we determined the effect of blocking E2F function on the development of hypertrophy. Fig. 5a shows that myocytes stimulated to undergo hypertrophy in the absence of any peptide or those exposed to the control peptides, AHS2 (30 µM) and ANT (30 µM), showed a significant induction of ANF and BNP, confirming that hypertrophic growth had occurred. However, cells stimulated to undergo hypertrophy with 20% FCS in the presence of AH2 (30 µM) showed a reduction in ANF and BNP levels compared with cells treated with control peptides. This reduction was even more pronounced in PE-stimulated cells wherein AH2 completely abrogated the induction of both ANF and BNP. Quantitative PCR analyses (Fig. 5b) showed that, following serum stimulation, the presence of AH2 inhibited ANF induction where levels were decreased 4-fold when compared with normal serum-stimulated cells and those with serum and control peptides, i.e. AHS2 and ANT. Similar to that seen with the semi-quantitative analyses, ANF induction was greatly reduced in AH2-treated cells (6-fold) when compared with PE-stimulated cells or PE-stimulated cells in the presence of control peptides (AHS2 and ANT) (Fig. 5c). Total protein content of myocytes also was compared in myocytes exposed to either 0% FCS or 20% FCS for 24 h in the presence or absence of E2F peptides (Fig. 6a). Thus, cells stimulated to undergo hypertrophy with 20% FCS doubled their total protein content over a 24 h period without any increase in cell number. AHS2- and ANT-treated cells also showed an approximate doubling of total protein content, indicating that the inclusion of a nonspecific exogenous peptide in the myocyte culture medium did not adversely affect the development of hypertrophy. In contrast, the addition of AH2 abolished the increase in total protein content in myocytes treated with 20% FCS such that levels remained similar to non-stimulated cells. There also was no significant change in cell number following treatment of myocytes with AH2 for 24 h, suggesting that the effect of this peptide was to inhibit hypertrophic growth. Similar effects were observed for PE-treated cells wherein AH2 substantially blocked the increase in total protein content that normally accompanies hypertrophic growth (Fig. 6b). The control peptides, AHS2 and ANT, had no such effects. This result suggests that blocking E2F function inhibits the protein synthesis that is known to occur during cardiac myocyte hypertrophy. In addition, AH2-treated myocytes did not show any increase in size 24 h post stimulation with 20% FCS, whereas cells treated with 20% FCS alone or 20% FCS plus AHS2 or ANT peptide approximately doubled in size (data not shown).
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DISCUSSION |
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Using an in vitro model of myocyte hypertrophy, we determined the expression patterns of E2F and DP family members during this process. Interestingly, a differential expression pattern of E2F and DP members was observed in myocytes undergoing hypertrophic growth such that there was a reversal toward the fetal pattern of protein expressions consistent with the transient up-regulation in specific cyclins and cyclin D-cyclin-dependent kinases during the development of left ventricular hypertrophy (4). E2F-1 expression levels were not changed significantly when myocytes were treated with PE, although a significant up-regulation was seen with FCS stimulation. Previously, E2F-1 has been implicated in cardiac myocyte cell cycle progression because adenoviruses expressing the E2F-1 gene caused a G1/S phase transition (13). However, cells did not progress further than the G2 phase of the cell cycle. The reason for the differential regulation of E2F-1 with serum compared with PE might reflect differences in signaling pathways (a growth factor-mediated pathway for FCS versus the 1-adrenergic receptor for PE) that mediate the hypertrophic response.
Our studies show that E2F-3a and, to a lesser extent E2F-4, are the predominant E2F species up-regulated both 24 and 48 h post stimulation with both hypertrophic agonists, suggesting an important role for these transcription factors in the hypertrophic growth of myocytes. It is possible that, during the development of hypertrophy, cardiac myocytes exhibit a temporal expression pattern of E2F as described by Takahashi et al. for human T98G glioblastoma cells (22). In this study, T98G cells were synchronized into the G0/G1 phase of the cell cycle by serum starvation and then stimulated to traverse the cell cycle by subsequent serum stimulation. The results showed that E2F-1 promoter activity was at its greatest 16 h post-stimulation and then declined, with E2F-3 taking a leading role late in the G1 phase when it bound to promoters for cyclin A, cdc2, cdc6, p107, and E2F-1, all of which are required for S phase progression. E2F-5 activity was not detected in these cells, possibly as a consequence of the fact that they were a proliferating population. It is possible that a similar scenario exists in cardiac myocytes such that E2F-1 activity could be dominant early on in hypertrophy and eventually become down-regulated to control levels, leaving E2F-3a to continue cell cycle progression and modulate growth of stimulated myocytes. Currently, E2F-4 is considered to be important for differentiation, along-side E2F-5 (7). However, we observed a significant presence of the E2F-4 protein in myocytes undergoing hypertrophy with both agonists. We therefore hypothesize that E2F-4 may be acting in conjunction with E2F-1 and E2F-3 to promote hypertrophy or that it could be removed from the nucleus following hypertrophic stimulation. Indeed, unlike the proliferative E2Fs (E2F-1, -2, and -3), both E2F-4 and E2F-5 contain nuclear export signals (NES) (7). Therefore, it is possible that E2F-4 is removed from the nucleus upon hypertrophic stimulation of the cardiac myocyte, thereby repressing its differentiation function.
In addition to determining the expression levels of the E2F proteins, we also investigated the global activity status of these transcription factors. Using a cyclin E promoter-luciferase assay, myocytes stimulated with 20% FCS showed a 2-fold increase in overall E2F activity, whereas cells stimulated with PE showed a 6-fold increase in overall E2F activity when compared with control cells (p < 0.05). These results strongly suggest that E2Fs play an important role in the development of hypertrophy following growth factor- or
1-adrenergic receptor-mediated stimulation. EMSAs confirmed these findings and showed an increase in E2F-DNA binding during the development of hypertrophy with serum and PE.
Inhibiting functional E2F activity with a specific peptide sequence that inhibits E2F-DP heterodimerization led to a significant inhibition in the development of myocyte hypertrophy as determined by a combination of ANF and BNP mRNA expressions and protein synthesis measurements. Both semi-quantitative (Fig. 5a) and quantitative PCR (Fig. 5b) analyses showed that blocking E2F function led to significant inhibition of hypertrophic growth, as levels of ANF and BNP were reduced to those seen in non-stimulated myocytes. Interestingly, semi-quantitative analysis showed that blocking E2F activity in PE-stimulated cells with AH2 has a greater effect, because ANF and BNP induction were reduced to undetectable levels (Fig. 5a). This finding is consistent with our luciferase assay results showing that PE-mediated hypertrophic growth is strongly E2F-dependant (Fig. 4a). Furthermore, inhibiting E2F function in 20% FCS-stimulated cells prevented protein synthesis, which doubled in cells exposed to FCS alone and in cells treated with FCS and the control peptides AHS2 or ANT. Other investigators have shown previously that targeting E2F can affect the growth of cardiovascular cells. Thus, Morishita et al. (24) showed that transfecting an E2F decoy oligonucleotide into rat vascular smooth muscle cells decreased proliferation of these cells and decreased the expression of E2F-dependent genes, namely, c-myc, cdc2, and the proliferating cell nuclear antigen. More recently, Mann et al. (10) used a similar approach to prevent neointimal hyperplasia in autologous vein grafts, a common problem that contributes to vein graft failure. Using an ex vivo pressure-mediated delivery of an E2F decoy oligonucleotide into veins, a transfection efficiency of 89% was achieved, and overall graft failure event rates fell to 29% compared with 69% for the control group. Clearly, blocking E2F function in the vascular system can provide significant clinical benefits for patients. In the present study, we have shown that blocking E2F function in cardiac myocytes may provide similar benefits for patients with hypertrophic cardiac disease.
In summary, we have shown that E2F and DP family members are regulated differentially during normal rat cardiac myocyte development such that all transcription factors, with the exception of E2F-5, are down-regulated as the heart matures. This pattern of expression is reversed during the development of myocyte hypertrophy, because E2F-1, -3a, and -4 and DP-1 are up-regulated, whereas E2F-5 is down-regulated following mitogenic stimulation. E2F activity is increased during the development of hypertrophy, and blocking E2F function inhibits the development of hypertrophy, suggesting that inhibiting E2F activity in certain cardiac diseases might provide a novel therapeutic approach for inhibiting the development of myocyte hypertrophy that might delay, and possibly prevent, the onset of heart failure.
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FOOTNOTES |
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To whom correspondence should be addressed: Cardiovascular Research Group, School of Animal and Microbial Sciences, The University of Reading, P. O. Box 228, Whiteknights, Reading, Berkshire RG6 6AJ, UK. Tel.: 44-118-931-6363; Fax: 44-118-931-6562; E-mail: g.brooks{at}reading.ac.uk.
1 The abbreviations used are: FCS, fetal calf serum; BrdUrd, bromodeoxyuridine; RT, reverse transcription; ANF, atrial natriuretic factor; BNP, brain natriuretic peptide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EMSA, electromobility shift assay; PE, phenylephrine; AH2, peptide sequence targeted to the DEF box region of the E2F heterodimerization domain; AHS2, scrambled sequence based upon AH2; ANT, penetratin sequence.
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REFERENCES |
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