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Cardiovascular Research 2003 59(2):512-519; doi:10.1016/S0008-6363(03)00392-4
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Copyright © 2003, European Society of Cardiology

Mechanism of E2F1-induced apoptosis in primary vascular smooth muscle cells

Jens Stanelle1, Thorsten Stiewe1,2, Florian Rödicker, Karin Köhler3, Carmen Theseling and Brigitte M. Pützer*

Center for Cancer Research and Cancer Therapy, Institute of Molecular Biology, University of Essen Medical School, Hufelandstrasse 55, 45122 Essen, Germany

* Corresponding author. Tel.: +49-201-723-3687; fax: +49-201-723-5974. brigitte.puetzer{at}uni-essen.de

Received 15 October 2002; accepted 1 April 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusion
 References
 
Objective: The transcription factor E2F1 serves as a major regulator of the cell-cycle by controlling G1-S phase transition. However, apart from its proliferative function high levels of deregulated E2F1 are capable of inducing apoptosis depending on the cellular context. In particular the tumor suppressor p53 and its homologue p73 are implicated in this proapoptotic function. Methods: Here, we investigated the mechanistic basis for E2F1-mediated apoptosis in vascular smooth muscle cells (VSMCs) which have previously been shown to be E2F1-responsive. Results: Interestingly, E2F1-expression in these cells induced clear signs of apoptosis in the absence of any proliferative activity. Although cell-cycle regulated genes such as CCNE1 and CDC25A were activated, BrdU-staining revealed no S-phase entry. Instead, a rapid loss of cell viability by induction of apoptosis was observed. Using a transactivation-defective E2F1-mutant, we show that apoptosis induction is independent of the transactivation function and therefore independent of ARF and p73. However, this mutant retains its ability to stabilize and phosphorylate p53, suggesting that p53 is sufficient for the effect of E2F1. Conclusion: VSMCs therefore represent a cellular system in which the transactivation-independent, proapoptotic activity of E2F1 is the primary cellular function. Ectopic expression of E2F1 might therefore be a suitable therapy to prevent VSMC hyperproliferation.

KEYWORDS Ad, adenovirus; ARF, ADP-ribosylation factor; MEF, mouse embryo fibroblast; OHT, hydroxytamoxifen; FACS, fluorescence-activated cell sorting; RT-PCR, reverse transcription polymerase chain reaction


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 5. Conclusion
 References
 
Over the past decade, a large number of studies revealed the central role of the RB-pathway in the regulation of G1/S transition and the control of cell proliferation by modulating the activity of the transcription factor E2F. From these studies it has become clear that E2F determines whether or not a cell will divide by controlling the expression of S-phase genes that encode cell cycle regulatory functions and DNA replication activities [1,2]. Despite the clear importance in allowing cell cycle progression, several studies have shown that particularly E2F1 promotes apoptosis in several systems [3–7]. Induction of apoptosis by either the loss of RB or the deregulation of E2F activity occurs both in association with p53 and independent of p53.

Ectopic expression of E2F1 has been shown to lead to increased levels of p53 [8], as a result of E2F1-mediated induction of the CDKN2A transcript p14ARF that in turn blocks MDM2-associated degradation of p53 [9]. Moreover, E2F1 can signal p53 phosphorylation that is coincident with p53 accumulation and apoptosis in the absence of ARF [10], similar to the observed stimulation of the apoptotic function of p53 in response to DNA damage by direct binding to E2F1 [11]. E2F1-induced apoptosis occurs also independent of p53 in tissue culture and transgenic mice [6,7,12,13], and RB has been shown to protect p53-null cells from apoptosis in an E2F1-binding dependent manner [14]. Mapping studies revealed that this ability of E2F1 requires its DNA-binding domain but not its transactivation function [6,7,15], suggesting that proapoptotic E2F1 target genes are activated by removal of E2F1/RB repression rather than direct transactivation [6,7,16]. However, we and others have recently identified the p53-homologue p73 as a target of p53-independent apoptosis [17,18]. E2F1 regulates p73 levels directly, through recognition and transactivation of the TP73 promoter. Recently, the gene for apoptosis protease-activating factor 1, Apaf-1, has been identified as another target by which E2F1 can induce apoptosis directly and independently of p53 induction [19]. Moreover, it is known that E2F1 expression can lead to the sensitization of cells to apoptosis independently of p53 by a death receptor-dependent mechanism in response to tumor necrosis factor {alpha} (TNF{alpha}) by downregulating TRAF2 protein levels and inhibiting antiapoptotic signaling such as NF-{kappa}B [20].

In the vascular system, E2F decoy oligonucleotides inhibit in vivo proliferation of human coronary vascular smooth muscle cells (VSMCs) and formation of postinjury neointima in balloon-injured rat carotid arteries [21]. Shelat et al. reported that overexpression of E2F1 in VSMCs leads to S-phase entry [22], followed by caspase 3-activation and apoptotic cell death. In contrast, restoration of E2F expression rescues vascular endothelial cells from TNF-{alpha} induced apoptosis [23]. In view of the central role of E2F1 in the regulation of cell growth and death in primary vascular cells, we analyzed the mechanism of E2F1-induced apoptosis in VSMCs.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 5. Conclusion
 References
 
2.1 Cell culture and virus construction
Passage-2 of human coronary VSMCs were purchased from Cascade Biologics, and were not used after passage 5. Cells were maintained in Dulbecco's modified Eagle medium (DMEM) or Medium 231 (Cascade Biologics) supplemented with 10% fetal bovine serum (FBS) or growth supplement SMGS (Cascade Biologics), respectively. All adenoviruses were grown in 293 cells (Ad5 E1-transformed human embryonic kidney cells) maintained in modified Eagle's medium (MEM) F-11 with 10% fetal bovine serum. Media were supplemented with 2 mM L-glutamine, 100 µg/ml penicillin, and 100 U/ml streptomycin. Adenoviruses encoding ER-E2F1, green fluorescent protein (GFP), and p53 have been described previously [24,25]. For generation of AdER-E(-TA), the cDNA fragment encoding the influenca hemagglutinin (HA)/estrogen receptor ligand binding domain (ER) chimaeric protein fused to the N-terminus (amino acid 1 to 374) of the E2F1 cDNA which lacks the transactivation domain between was cloned into pMH4. Recombinant adenovirus was produced as described [24].

2.2 Western blotting
For Western blot analysis, cells were infected at a multiplicity of infection (MOI) of 100 plaque forming units (PFU) per cell. At 72 h after infection, cell lysates were prepared and protein levels were analyzed essentially as described [17]. Samples were probed with mouse anti-human E2F1 monoclonal antibody (KH95, Santa Cruz Biotechnology), murine anti-p53 monoclonal antibody (DO-1, Calbiochem) or phospho-p53 (Ser15) antibody (Cell Signaling Technology). Full-length caspase-3 (35 kDa) and its large cleavage product (17 kDa) was detected using antibodies directed against Caspase-3 (9662) and Cleaved Caspase-3 (9661; Cell Signaling Technology). Antibody binding sites were visualized using appropriate horseradish-peroxidase conjugated secondary antibodies according to the enhanced chemiluminescence (ECL) protocol (Amersham).

2.3 Cell viability assay
For MTT cell viability assays, serum-starved cells were infected and cell viability was determined in the presence and absence of OHT at a final concentration of 1 µM over 3 days after infection. Triplicate wells of each treatment were assayed for cell viability by the CellTiter96® AQueous One Solution Cell Proliferation Assay (Promega).

2.4 Apoptosis assay
To visualize apoptosis in unfixed monolayer cultures, 72 h after infection the cells were incubated at 37°C in the presence of 1 µg/ml Hoechst 33342. 15 min later propidium iodide (PI) solution was included to 5 µg/ml and monolayers were observed by epifluorescence microscopy [26]. Cells dissociating from the monolayer exhibited bright blue, fluorescing masses of chromatin that abutted at the nuclear membrane or, at later stages showed bright blue, fluorescing spherical bodies and were therefore identified as apoptotic.

2.5 DNA fragmentation assay
For low-molecular-weight DNA extraction, VSMCs were infected with AdER-E2F1 or AdER-E(-TA) in the presence of OHT for 72 and 96 h. Cells were lysed in 50 mM Tris, pH 7.8, 10 mM EDTA, 1% sodium dodecyl sulfate (SDS), and 0.5 mg/ml proteinase K. After overnight incubation at 37°C, lysates were digested with 5 µg/ml RNase A for 3 h at 37°C, extracted twice with phenol–chloroform–isoamyl alcohol and DNA was precipitated overnight. Samples were analyzed by electrophoresis in 1.5% agarose gels in TBE buffer (90 mM Tris, 90 mM Boric acid, 2 mM EDTA (pH 8.0)). Analysis was performed with DNAs extracted from equal numbers of cells.

2.6 RT-PCR analysis
Semiquantitative RT-PCR was performed on total RNA from serum-starved cells infected with Ad vectors and prepared by RNeasy Mini Kit (Qiagen, Hilden, Germany). PCR amplification was performed as described [17]. A minimum amount of cycles was carried out to stay within the linear amplification process. Primer sequences are available upon request.

2.7 Flow cytometry
To quantitate apoptosis, serum-starved cells were infected at 60–80% confluence and further incubated in the absence or presence of 1 µM OHT. Cells were harvested 72 h after infection, fixed in 70% ethanol and stained for DNA content with propidium iodide. For analysis of S-phase entry, VSMCs were labeled with 5-bromo-2'-desoxyuridine (BrdU) 24 h after infection using the In Situ Cell Proliferation Kit FLUOS (Roche). BrdU incorporation was detected using fluorescein isothiocyanate (FITC)-linked anti-BrdU according to the manufacturer's protocol. Flow cytometric measurements were performed in a FACSVantage sorter (Becton Dickinson) and analyzed using CellQuest software (Becton Dickinson).


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 5. Conclusion
 References
 
3.1 E2F1-mediated transactivation of target genes
There is evidence that ectopic expression of the E2F transcription factor can induce both S-phase progression and apoptosis, depending on the cell type context [6–8,27,28]. We have previously developed an inducible Ad vector system, in which regulation of potentially cytotoxic gene products such as E2F1 is achieved by fusion of the transgene to the OHT regulatable ER domain [24]. By using this vector in the presence of OHT, we have shown that E2F1 can be efficiently activated in quiescent fibroblasts and several human tumor cell lines, resulting in the induction of genes involved in cell cycle progression and apoptosis [24,29]. To analyze the transactivation function of E2F1 in primary VSMCs, we infected serum-starved cells with AdER-E2F1 or a transactivation-defective E2F1-mutant, AdER-E(-TA) in the absence or presence of OHT and measured the expression of three known direct E2F target genes by semiquantitative RT-PCR. Activation of E2F1 led to increased mRNA levels of CCNE1 (encoding cyclin E) [30], CDC25A encoding CDC25A phosphatase, shown to be required for efficient E2F1-induced S-phase [31], and TP73 which encodes the proapoptotic p53-homologue p73 [17] at 8 h after induction (Fig. 1). In contrast, the transactivation-defective mutant E(-TA) did not induce target gene expression. Also no effect was detectable in virus-infected VSMCs without OHT and in the GFP control. These data demonstrate that E2F1 is competent as a transcriptional activator in VSMCs, whereas mutant E2F1 is not.


Figure 1
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  		Fig. 1 Transactivation of E2F1 target genes. Semiquantitative RT-PCR analysis on total RNA from VSMCs infected with AdER-E2F1, AdER-E(-TA), and AdGFP in the absence or presence of OHT for CCNE1, CDC25A, TP73, and GAPDH expression was carried out under linear amplification conditions. GAPDH expression is shown as a control. One representative experiment of three is shown.

 
3.2 Block of S-phase entry
Next, we examined whether E2F1 gene transfer promotes S-phase entry in growth arrested VSMCs. Following 2 days of serum starvation, VSMCs were infected with adenovirus expressing either E2F1 or E(-TA) and labeled with BrdU at 24 h after infection. BrdU incorporation was determined by immunofluorescence. In response to serum, cells showed a rapid entry into S-phase (Fig. 2, left panel). Interestingly, however, very little S-phase entry was observed in VSMCs infected with AdER-E2F1 or AdER-E(-TA) in the presence of OHT where the percentage of cells in the S-phase remained consistently<5% (Fig. 2, middle and right panel). Thus, E2F1 does not lead to S-phase entry in VSMCs.


Figure 2
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  		Fig. 2 E2F1 does not lead to S-phase entry in VSMCs. Growth-arrested cells were infected with Ad vector expressing E2F1 or E(-TA) in the presence of OHT. At 24 hours post infection cells were labeled with BrdU and DNA synthesis was determined by flow cytometry. Compared to serum-induced cells showing S-phase entry (left panel), the percentage of cells in the S-phase after AdER-E2F1 (middle panel) or AdER-E(-TA) (right panel) infection remained consistently<5%. One representative experiment of three is shown.

 
3.3 Apoptosis induction is independent of the transactivation function
To directly assess the cytotoxic effect of E2F1 in VSMCs, growth-arrested cells were incubated with AdER-E2F1, AdER-E(-TA), and AdGFP control virus in the presence of OHT over 3 days. Cytotoxicity was analyzed by quantitating viable cells using the MTT assay. As shown in Fig. 3A, overexpression of both E2F1 and the mutant E2F1 protein [E(-TA)] resulted in a substantial loss of cell viability by more than 90% at day 3, compared to control vector (mock) infected VSMCs (~40%). Within 36 to 72 h after Ad vector-mediated gene transfer of E2F1 and E(-TA) to VSMCs, morphological changes characteristic for cells undergoing apoptosis were observed with cells rounding up, membrane blebbing, cell shrinkage, and condensation of chromatin (Fig. 3B). In support, flow cytometry analysis of serum-starved VSMCs infected either with AdER-E2F1 or AdER-E(-TA) in the presence of OHT revealed a significant increase of sub-G1 cells on day 3 (Fig. 4AIII, V) of 43.5% (E2F1) and 49.9% [E(-TA)], respectively, indicative of apoptosis. In contrast, in the absence of tamoxifen or in mock infected cells, no significant increase in the sub-G1 population was observed (Fig. 4A, I, II and IV). Induction of apoptosis in VSMCs by E2F1 and E(-TA) was also accompanied by processing of caspase-3, as revealed by the appearance of a ~17 kDa product which corresponds to the 17 kDa subunit of activated caspase-3 (Fig. 4B). As shown in Fig. 4C, a typical DNA laddering pattern consistent with apoptosis was evident at 72 and 96 h after infection with AdER-E2F1 (lanes 1 and 3) as well as AdER-E(-TA) (lanes 2 and 4). These data indicate that E2F1 clearly induces apoptosis in VSMCs which is independent of the transactivation function.


Figure 3
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  		Fig. 3 Cytotoxic effect of E2F1 in VSMCs. VSMCs infected with AdER-E2F1 or AdER-E(-TA) and induced by OHT show a substantial loss of cell viability within 3 days after infection. (A) MTT cell viability assay. AdER-E2F1 ({blacksquare}), AdER-E(-TA) (bullet) and AdGFP ({circ}). Shown is the mean of three independent experiments±S.D. Significant differences (P<0.001; paired, two-sided t-test) between AdER-E2F1, AdER-E(-TA), or AdGFP and untreated cells (set as 1) are labelled with *. (B) Morphologic changes of VSMC expressing ER-E2F1 or ER-E(-TA) compared to Mock-infection are shown by laser scan microscopy (upper panel) and Hoechst 33342-fluorescence micrographs (lower panel). Infected cells dissociating from the monolayer exhibited bright blue fluorescing masses of chromatin that abutted at the nuclear membrane and were therefore identified as apoptotic (marked by arrows).

 

Figure 4
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  		Fig. 4 E2F1-mediated apoptosis is independent of the transactivation function. (A) Serum-starved primary vascular smooth muscle cells (I) were infected with AdER-E2F1 (II, III) or the transactivation-defective E2F1 mutant (IV, V) in the absence (II, IV) or presence (III, V) of OHT. At 72 hpi cells were harvested and processed for PI staining and flow cytometry. A significant cell population in sub-G1 was seen in both AdER-E2F1 (43.5%) and AdER-E(-TA) (49.9%) infected cells with OHT. One representative experiment of three is shown. (B) Activation of caspase-3 in cells infected with AdER-E2F1 or AdER-E(-TA) over 5 days was analyzed by Western blotting. Full-length caspase-3 (35 kDa) and the cleaved 17 kDa subunit are indicated. (C) DNA fragmentation in VSMCs overexpressing E2F1 and E(-TA) detected by gel electrophoresis. DNA fragmentation in the form of oligonucleosomal DNA ladders occurred in growing VSMCs in the presence of OHT at 72 (lanes 1 and 2) and 96 h after infection (lanes 3 and 4) with AdER-E2F1 (lanes 1 and 3) and AdER-E(-TA) (lanes 2 and 4, respectively). Lane M, small fragment DNA ladder; lane 0, untreated cells at 96 h.

 
3.4 p53 accumulation and phosphorylation by E2F1
We and others have previously shown that part of the p53-independent apoptotic activity of E2F1 reflects its ability to induce p73 expression [17,18] and that the mechanism of induction depends on the transactivation domain ([18], unpublished data). As shown in Fig. 1, the E2F mutant which lacks the transactivation domain is unable to stimulate p73 expression in VSMCs. Because the E2F mutant is capable of inducing apoptosis at levels similar to the wild-type transcription factor, it appears that apoptosis induction by E2F1 is independent of p73. We therefore examined the contribution of p53 to E2F1-induced apoptosis in these cells. VSMCs were infected with AdER vector expressing E2F1 or E(-TA), respectively, in the absence or presence of OHT, and cell extracts were subsequently analyzed for p53 protein levels. Consistent with the apoptotic rate measured by flow cytometry, both E2F1 and the transactivation-defective mutant induced equally high levels of p53 compared to the controls (Fig. 5), indicating that the E(-TA) mutant retains its ability to stabilize p53. Based on previous findings, there are different pathways for E2F1 to signal p53-mediated apoptosis and p53 accumulation. Since E2F1-mediated activation of p53 via the ARF/Mdm2 pathway requires the transactivation domain of E2F1, it is likely that p53 stimulation occurs through a ARF-independent mechanism. Therefore, we tested the ability of E2F1 to induce a change in p53 phosphorylation. Importantly, we found that ectopic expression of both E2F1 and E(-TA) in VSMCs resulted in increased p53 phosphorylation of serine 15, which has recently been shown to contribute to E2F1-mediated apoptosis in the absence of ARF [10] (Fig. 5). These data suggest that this covalent modification of p53 is sufficient for E2F1-mediated apoptosis in VSMCs.


Figure 5
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  		Fig. 5 p53 expression and phosphorylation by E2F1. Western blot analysis for endogenous p53, the phospho-Ser15 form of p53, and E2F1 in lysates of human VSMCs. Cells were infected with AdER-E2F1 and AdER-E(-TA), respectively, in the absence or presence of OHT, or the control vector AdGFP. Uninfected cells are shown as a control (mock). One representative experiment of two is shown.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
5. Conclusion
 References
 
During normal cell proliferation, E2F modulates the expression of many genes involved in G1-S phase transition and DNA replication [2]. In addition to the well-established proliferative effect, particularly E2F1 has also been implicated in the induction of apoptosis [5] by both p53-dependent and p53-independent pathways [6,7,13,27,28,32,33]. In agreement with these studies, it has been shown that E2F1 overexpression mediates growth suppression in coronary VSMCs which involves caspase 3-like activity [22].

Here, we have investigated the mechanism of E2F1-induced cell death in primary vascular smooth muscle cells. We found that increased E2F1 activity results in a rapid loss of cell viability by the induction of apoptosis in VSMCs. In contrast to a previous work [22], however, suggesting that E2F1 regulates growth of VSMCs by forcing the cells to enter the S-phase and then to die, we show that E2F1 is unable to induce S-phase entry in growth-arrested cells. Thus, the consequence of increased E2F1 activity in VSMCs is apoptosis, and not DNA replication. Nevertheless, E2F1 activation leads to increased expression of cell cycle regulated target genes (as shown here for CCNE1 and CDC25A) in the absence of S-phase entry, demonstrating that E2F1 is competent as a transcriptional activator in vascular smooth muscle cells. Our results agree with previous findings indicating that E2F1 is not sufficient to induce S-phase progression in quiescent human diploid fibroblasts unless other genetic alterations (transformed cells) occur [34,35]. In the absence of proliferative E2F activity, Lomarri et al. also observed increased expression of S-phase relevant genes such as CCNE1 after E2F1 activation [35]. Based on their findings, E2F1-induced S-phase entry requires suppression of the RB- or p53-regulated G1 checkpoint, which is consistent with the observation that p53 and RB negatively regulate the cell cycle of primary VSMCs [36]. By contrast, E2F1 efficiently induced cell cycle progression in cells that are impaired in RB- or p53 function [37]. At the G1 checkpoint, RB acts as a transcriptional repressor of E2F1 by preventing it from S-phase entry [38].

Our results, however, indicate that both E2F1 and the mutant lacking the transactivation domain induce similar levels of apoptotic cells, suggesting that apoptosis induction by E2F1 is independent of the transactivation function. Since E2F1-mediated activation of p53 via the ARF/Mdm2 pathway requires the transactivation domain of E2F1, it is therefore likely that p53 stimulation in vascular smooth muscle cells occurs through a ARF-independent mechanism. Interestingly, we have seen increased p53 phosphorylation by ectopic expression of both E2F1 and the transactivation-deficient mutant. This result seems mechanistically similar to a previous work showing that E2F1 can signal p53 phosphorylation in the absence of ARF in mouse embryo fibroblasts [10]. From these data, it appears that the covalent modification of p53 which is coincident with p53 accumulation contributes to E2F1-mediated apoptosis in VSMCs.

We and others have previously shown that activation of p73 provides a means for E2F1 to induce cell death independent of p53 [17,18]. Disruption of p73 function inhibits E2F1-induced apoptosis in p53–/– MEFs. Whereas activation of p53 in response to E2F1 is indirect involving ARF, E2F1 regulates p73 levels directly through recognition and transactivation of the TP73 promoter [17,18]. E2F1-mediated transactivation of p73 then results in the activation of p53-responsive target genes and apoptosis. This is again demonstrated in VSMCs, where the E2F mutant which lacks the transactivation domain is unable to stimulate p73 expression, but is capable of inducing apoptosis at levels similar to the wild-type transcription factor. From these data, we conclude that apoptosis by E2F1 in VSMCs is also independent of p73. VSMCs therefore seem to represent a cellular system, where the transactivation-independent, apoptotic activity of E2F1 is the primary cellular function.


   5. Conclusion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusion
References
 
Increased E2F1 activity can induce apoptotic cell death in VSMCs, in the absence of S-phase entry. Apoptosis induction by E2F1 is independent of its transactivation domain and does therefore not require ARF or p73, suggesting that VSMCs represent a cellular system in which the transactivation-independent, proapoptotic activity of E2F1 is the primary cellular function. Instead, increased p53 expression and phosphorylation appear to be crucial for E2F1-mediated effect in VSMCs. Thus, overexpression of E2F1 may provide a suitable gene therapeutic strategy to prevent VSMC hyperproliferation in atherosclerosis, hypertension, and restenosis after injury.

Time for primary review 24 days.


   Acknowledgements
 
This work was supported by the IFORES program of the Medical Faculty of the University of Essen.


   Notes
 
1 Both authors have contributed equally to the data presented. Back

2 Present address: Rudolf-Virchow-Zentrum, University of Würzburg, Versbacher Strasse 9, 97078 Würzburg, Germany. Back

3 Present address: Department of Internal Medicine, West German Cancer Center, University of Essen Medical School, Hufelandstrasse 55, 45122 Essen, Germany. Back


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusion
 References
 

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