Department of Cell Biology, Institute of Anatomy and Cell Biology, Göteborg University, Box 420, SE-405 30 Gothenburg, Sweden
¶ Author for correspondence (e-mail: keiko.funa{at}anatcell.gu.se)
Accepted 17 March 2004
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Summary |
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Key words: Platelet-derived growth factor, ß-receptor, SV40LT, pRb, c-Myc, p53
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Introduction |
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It has previously been reported that c-Myc represses PDGF ß-receptor expression at the transcriptional level through its interaction with NF-Y (Izumi et al., 2001; Oster et al., 2000
). We have recently demonstrated that c-Myc interacts with p73 (Uramoto et al., 2002
), which, independently of c-Myc, also represses transcription of PDGF ß-receptor (Hackzell et al., 2002
). Similar mechanisms seem to prevail in the repression exerted by c-Myc and p73
through their interaction with the NF-Y transcription factor. NF-Y binds the consensus CCAAT motif in the PDGF ß-receptor promoter, and controls its basal activity together with Sp1 that binds in close proximity (Ballagi et al., 1995
; Ishisaki et al., 1997
; Molander et al., 2001
). NF-Y consists of NF-YA, NF-YB and NF-YC subunits that are all necessary for DNA binding (Kim et al., 1996
). c-Myc and p73
bind the C-terminal HAP domain of NF-YB and NF-YC and inactivate the transcription.
Previously it has been shown that the large T antigen (LT) and small t antigen of simian virus 40 (SV40) down-regulated the expression of both types of PDGF receptor in various kinds of fibroblasts (Wang et al., 1996). They mainly studied the PDGF
-receptor downregulation at the mRNA level, which was shown to occur independently of p53 and Rb. However, the mechanism behind the PDGF ß-receptor repression by LT and whether c-Myc, p73
, or as yet undiscovered molecules are involved in the mechanism, still remains unresolved.
SV40 was first isolated in Rhesus monkey cells used to grow the active polio vaccine developed in the late 1950s (Hilleman, 1998). The virus belongs to the polyoma virus family and has been extensively studied as a model to investigate the control mechanism of cell growth (for a review, see Weiss et al., 1998
). In addition, the involvement of SV40 in human tumours was recently verified by the identification of the viral sequence in certain tumour tissues (Klein et al., 2002
), emphasising the importance of understanding the molecular mechanism of SV40 in tumour pathogenesis. The LT, a 90-kDa phosphoprotein, is the only viral protein essential for SV40 replication.
The LT was shown to be a molecular chaperone, promoting the proper folding of proteins and preventing protein aggregation during cellular stress (Hartl and Martin, 1995). The chaperone activity is necessary for viral replication, transcriptional control, virion assembly, and transformation (Sullivan and Pipas, 2002
). The LT has several functions, including control of the activities of ATPase, DNA-binding, oligomerisation and DNA helicase. Following infection, LT affects host gene expression and growth control by binding to a wide variety of transcription factors that are important for both replication and cell cycle regulation including tumour suppressors, p53 and retinoblastoma family proteins (Moens et al., 1997
). These interactions are thought to play crucial roles in the pathogenesis and progression of tumours.
The retinoblastoma protein (pRb) prevents cell cycle progression by binding and sequestering E2F (Classon and Harlow, 2002). It has been shown that the E2FRb complex can also directly repress the promoter of certain cell cycle genes by recruiting histone deacetylase (Magnaghi-Jaulin et al., 1998
). The N-terminal LT binds the E2F-Rb complex and dissociates it and thereby preventing the repression (Laufen et al., 1999
). One of the activated downstream target genes is c-myc (Batsche et al., 1994
), which is necessary to induce and maintain proliferative cell states (for a review, see Facchini and Penn, 1998
). In this manner, SV40 keeps host cells in a proliferating state to use them for its own replication. Another critical control mechanism of SV40 on host replication is through the interaction of the C-terminal LT with the p53 tumour suppressor (Kierstead and Tevethia, 1993
). The aim of this study is to clarify the mechanisms that LT uses to repress PDGF ß-receptor expression by using normal 3T3 fibroblasts and cell lines deficient in c-Myc, pRb, p53 or NF-Y and Sp1.
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Materials and Methods |
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Plasmid constructs
The cDNAs for SV40LT wild type (WT) and the mutant K1 (E107K) containing an amino acid substitution at position 107 in the pRb-binding motif in pCDNA3 were provided by Dr Livingston (Dana-Faber Cancer Institute, Boston, MA). The cDNAs for the mutant C105G, containing an amino acid substitution at position 105 in the pRb-binding motif (C105G), the mutant 434-444, lacking the p53-binding motif and the H42Q with an amino acid substitution at position 42, inactivating the J domain function of LT in pSG5 expression vector, were provided by Dr DeCaprio (Chao et al., 2000
). The J domain is thought to assist pRb binding. The expression levels of all the constructs were examined and the expression of LT in two different vectors was judged to be similar by immunoblotting 3T3 cell lysates after transfection. For functional promoter assays, the promoter region of mouse PDGF ß-receptor was inserted in a luciferase expression vector, pGL3 (Promega) (Ishisaki et al., 1997
). The dominant negative mouse NF-YA (DNNFYA) was provided by Dr Mantovani (Mantovani et al., 1994
), and Sp1 cDNA was provided by Dr Tjian (Howard Hugh Medical Institute, University of California, CA). The pPacSP1 and pPacNF-Y expression vectors were provided by Dr Suske (University of Marburg, Germany) and Dr Zetterberg (Göteborg University, Sweden).
Receptor binding assay
To estimate the relative amounts of functional PDGF receptor in the presence or absence of LT expression, we used the ST15A cell line. The amount of PDGF receptor binding was determined by analysing serial dilutions of cold PDGF ligand with regard to its ability to compete with [125I]PDGF ligand for binding to the cells. Cells were grown on 24-well plates (Becton Dickinson) at 33°C, or first at 33°C and then at 39°C for at least 5 days before being used for the assay. For the ß-receptor assay the -receptor was depleted by a 60-minute preincubation with 50 ng/ml PDGF-AA at 37°C. Cell cultures were washed once in binding buffer (phosphate buffered saline, PBS, containing 1 mg/ml bovine serum albumin, BSA, 0.9 mM CaCl2, and 0.5 mM MgCl2) and then incubated at 0°C for 2 hours in 200 ml binding buffer containing various dilutions of PDGF-BB. The cells were washed in binding buffer before addition of the labelled ligand (0.5-2 ng containing 15,000-30,000 cpm). After incubation at 0°C for 1 hour, the cells were washed with binding buffer, then lysed in 200 ml of 20 mM Tris-HCl, pH 7.5, 1% Triton X-100 and 10% glycerol at room temperature for 20 minutes. The amount of solubilised [125I] radioactivity was measured in a
-counter.
Immunohistochemistry
For immunohistochemical staining of PDGF ß-receptor, ST15A cells cultured on chamber slides (Nunc) at 33°C or 39°C, as described above, were fixed in cold 4% paraformaldehyde in PBS. They were stained with the anti-rat PDGF ß-receptor (Ab-1; Oncogene Science) using an ABC immunoperoxidase method essentially as described (Funa et al., 1996). The fixed cells were incubated with 20% normal goat serum, 2% normal rat serum and 2% BSA in PBS for 10 minutes. Incubation with the primary antibody diluted 1:500 in PBS was performed for 1 hour at room temperature. Following incubation with the biotinylated anti-rabbit Ig, the immunocomplex was coupled to ABC Elite complex (Vector) which was visualised with 3-amino-9-ethylcarbazole as the chromogen and 0.02% H2O2 as substrate. The slides were counterstained with haematoxylin and mounted in glycerol-gelatin. A negative control for each slide was performed by substituting the primary antibody with 1% BSA in PBS. Cells transfected with a mouse PDGF ß-receptor expression vector or the empty vector was used as positive or negative controls, respectively.
Transient plasmid transfection and immunoblotting
NIH3T3 cells and HO15.19 cells in 10 cm dishes were transiently transfected with 12 µg of expression plasmids using 60 µl Fugene (Roche). Cells were transfected with LT, small t or expression vector, and cultured with 10% serum. After 48 hours cells were collected and lysed with 50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 150 mM NaCl, 1% Triton-X100, 0.05% sodium dodecyl sulfate (SDS), and 1 mM phenylmethylsulfonyl fluoride (PMSF). Cell lysate, 200 µg, was separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto immobilon-P membrane filters. After immunoblotting with anti-PDGF ß-receptor (958; Santa Cruz), anti-LT (Pab 108; Santa Cruz), or anti-Sp1 (1C6, Santa Cruz) antibodies, the membranes were developed by the enhanced chemiluminescence (ECL) protocol (Amersham). Wild type and Rb/ 3T3 cells were serum-depleted in DMEM with 0.5% FCS for 48 hours before being stimulated with 10% FCS for the indicated time periods. Cells were harvested and immunoblotted with anti-PDGF ß-receptor, anti-c-Myc (9E10, Santa Cruz), and anti-actin (AC-40, Sigma) antibodies. Total cell lysate from ST15A cells was extracted from cells cultured at 33°C and 39°C, respectively, and immunoblotted with anti-LT and p53 (DO-1, Santa Cruz) antibodies as described above.
Reverse Transcription (RT)-PCR
3T3 cells were seeded in 6-cm dishes at a density of 1.1x105 cells/dish. After 24 hours, half of the cells of each cell type were transfected with 2 µg of LT expression plasmid/dish and the remaining cells were transfected with 2 µg of the vector plasmid alone. The cells were kept in 10% FCS, and RNA was extracted essentially as described (Chomczynski and Sacchi, 1987) at 0, 2, 4 and 8 hours after transfection. 3 µg of total RNA from each time point was transcribed into cDNA with random primers and Moloney murine leukaemia virus reverse transcriptase (Invitrogen) according to the manufacturer's protocol. PCR was performed by using Taq polymerase (Fermentas) and a programmable thermal block (LabLine). PDGF ß-receptor and ß-actin primers were used as described previously (Ballági-Pordany et al., 1991
). PCR products were identified by dot-blot hybridisation, and analysed on 1.5% TAE-agarose gel stained with ethidium bromide for comparison.
Promoter reporter assay
Cells were seeded in 12-well plates at a density of 2x104 cells/well in 10% FCS. The following day, the cells were transiently transfected with 0.2 µg reporter plasmid, 0.25 to 0.5 µg of expression plasmid. Each expression plasmid and reporter plasmid was standardised individually using a molar ratio of 1:1 and the total amount of DNA per well was adjusted to 1.0 µg by addition of mock DNA plasmid. Sp1 was used as a positive control. After 48 hours cells were lysed with 100 µl/well of reporter lysis buffer (Promega). Luciferase activity was measured according to the vendor's instruction (Promega). Standardisation by co-transfection with ß-galactosidase reporter plasmid was avoided because LT influences expression of most promoters of plasmids used as internal controls (Moens et al., 2001). Results shown were thus normalised to protein concentration and were representative of at least three independent experiments.
Chromatin immunoprecipitation (Chip) assay
Chip assay was performed as previously described (Uramoto et al., 2002). Briefly, protein and DNA were cross-linked by incubating ST15A cells with formaldehyde at a final concentration of 1% for 10 minutes at room temperature. Cells were then lysed in Buffer X (50 mM Tris-HCl at pH 8.0, 1 mM EDTA, 120 mM NaCl, 0.5% NP-40, 10% glycerol, and 1 mM PMSF) for 15 minutes on ice. The lysate was sonicated and soluble chromatin was pre-cleared by addition of 10 mg Protein A-sepharose. An aliquot of pre-cleared chromatin was removed and used in the subsequent PCR analysis. The remainder of the chromatin was diluted with Buffer X. Then protein-DNA was incubated with 2 µg LT antibody, p53 antibody (DO-1), NF-YB (FL-207X, Santa Cruz), normal rabbit serum or mouse IgG in a final volume of 800 µl overnight at 4°C. Immune complexes were collected by incubation with 15 µl Protein G-agarose (Santa Cruz) for 1 hour at 4°C. Protein G-agarose pellets were washed once with 1 ml Buffer X, once with high salt Buffer X (500 mM NaCl), once with LiCl buffer (10 mM Tris, 1 mM EDTA, 0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, pH 8.1) and twice with TE (10 mM Tris, 1 mM EDTA, pH 8.0). Immune complexes were eluted twice with 250 µl of elution buffer (0.1 M NaHCO3, 1% SDS). To reverse protein-DNA cross-linking, eluted samples were incubated with 0.2 M NaCl for 4-5 hours at 65°C. Samples were digested with Proteinase K (0.04 mg/ml) for 2 hours at 45°C and then with RNase A (0.02 mg/ml) for 30 minutes at 37°C. DNA was purified with phenol:chloroform followed by ethanol precipitation. Purified DNA was resuspended in 10 µl H2O. Aliquots of 2 µl serial dilution were analysed by PCR with the appropriate primer pairs. The proximal PDGF ß-receptor promoter (229/+273) primers were 5'-GGGAGGGAGCAGGAGGGAAAGGAG-3' and 5'-GAATCAGGGGAATGGAGAGGGTGC-3'. The distal PDGF ß-receptor promoter (1764/1424) primers were 5'-CCTCAGGTAGTCATGGTCTC-3' and 5'-TGCCAGACCACAGGATAATG-3'. Amplification was performed for a predetermined optimal number of cycles. PCR products were separated by electrophoresis on 2% agarose gels, which were stained with ethidium bromide.
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Results |
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Downregulation of PDGF ß-receptor expression by SV40LT transfection
We examined the level of PDGF ß-receptor protein expression by immunoblotting 3T3 cells following transfection of LT, small t, or the vector alone. Cells transfected with LT alone expressed a significantly lower level of PDGF ß-receptor, whereas cells transfected with small t showed only a slight decrease in the expression level (Fig. 2A). To see whether c-Myc is directly involved in the LT-induced repression of PDGF ß-receptor expression, we examined the c-myc/ HO15.19 fibroblast cell line. PDGF ß-receptor expression was high in this cell line (Oster et al., 2000) and transfection with LT did not alter the expression level (Fig. 2B). We examined the kinetics of the mRNA expression in NIH3T3 cell line at 0, 2, 4 and 8 hours after transfection with LT or a control vector by RT-PCR. PDGF ß-receptor mRNA, as judged by PCR products and in comparison with ß-actin mRNA, gradually decreased following the transfection of LT but not of a control vector (Fig. 2C).
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The CCAAT motif is important for SV40LT repression on PDGF ß-receptor promoter activity
We examined whether the LT downregulation of PDGF ß-receptor stems from the effect on the transcriptional activity, and if so, which area of the promoter is responsible for the repression (Fig. 3A). The SacI/SacI promoter reporter construct (pGL31481; 1481 to +18) contains a CCAAT motif located 62 bp upstream of the first initiation site and also the GC boxes that were shown to bind Sp1 (Molander et al., 2001) and the SacI/SacI mCCAAT with a mutation in the CCAAT motif (Ishisaki et al., 1997
). The MluI/SacI (122 to +18) also contains the CCAAT motif and the GC boxes, the HindIII/SacI (69 to +18) contains the CCAAT motif without a GC box (Ballagi et al., 1995
). As shown in Fig. 3B, co-transfection of a LT expression vector decreased the luciferase activity of the SacI/SacI PDGF ß-receptor promoter in a concentration-dependent manner when compared with that of a control vector alone. Small t decreased the activity only slightly as judged by repeated experiments. LT also decreased the activity of the HindIII/SacI as well as the MluI/SacI promoter constructs, indicating that the CCAAT motif may be needed for the repression to occur (Fig. 3C). Sp1 used as a positive control showed a strong increase when the major Sp1 binding GC-rich sequence located between the MluI and HindIII sites was present in the promoter reporter.
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In order to determine whether the CCAAT-binding transcription factor NF-Y is required for the LT mediated repression, we co-transfected the DNA-binding defective and thus dominant negative NF-YA (DNNFYA) (Mantovani et al., 1994) together with the LT expression vector and the SacI/SacI promoter luciferase plasmid (Fig. 3D). As expected, co-expression of 0.5 µg DNNFYA decreased the PDGF ß-receptor promoter activity to 51% of that obtained with vector alone. Co-expression of LT decreased the promoter activity obtained by transfection with control vector alone by 62%, whereas a promoter decrease of only 32% was seen when 0.5 µg DNNFYA construct was additionally transfected together with LT or the control vector. Sp1 used as a positive control increased the promoter activity about twofold even in the presence of DNNF-Y when compared with DNNF-Y without Sp1 co-transfection (Molander et al., 2001
). However, the increase was smaller than that obtained when Sp1 was transfected alone without DNNF-Y, which resulted in around a fourfold increase (Fig. 3C).
Effects of various SV40LT mutants on the PDGF ß-receptor promoter activity in 3T3 cells
Various LT mutants including the three pRb-binding defective mutants were examined in order to determine the domains of LT necessary to repress PDGF ß-receptor promoter activity in 3T3 cells. LT contains a four-helix bundle, residues from helices 2 and 4 called the J domain and a loop containing the LXCXE motif. The N-terminal J domain and this loop interact with the highly conserved A and B domains of pRb (Sullivan and Pipas, 2002). As shown in Fig. 4A, co-transfection of either one of the LXCXE motif mutants, K1 or C105G, caused more than a twofold increase of PDGF ß-receptor promoter activity compared with control vector. The J domain-inactivating mutant H42Q neither increased nor decreased the activity. The p53 binding mutant,
434-444, did not decrease the promoter activity, indicating that the p53-binding domain is also needed for the repression of the promoter. These findings suggest that the binding of LT to both pRb and p53 plays an important role in the repression.
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In contrast to the wild-type LT, co-transfection of the K1 mutant increased the activity, in a concentration-dependent manner, of both the SacI/SacI and the shorter HindIII/SacI constructs, as shown in Fig. 4B. Furthermore, a mutation in the CCAAT motif completely abolished the activation of the SacI/SacI construct by K1 (Fig. 4C). Co-transfection of DNNF-YA also abolished the activation of the wild type SacI/SacI by K1. These results indicate that the K1 activates the promoter through the NF-Y-binding site.
pRb, Myc, and p53 are required for repressing PDGFß-receptor promoter activity by SV40LT
In order to see whether the major targets of LT, pRb, and p53 are directly involved in the LT induced repression of PDGF ß-receptor promoter, we examined the promoter activity in the Saos-2 cell line lacking both pRb and p53 (Fig. 5A). As expected, LT could not alter the PDGF ß-receptor promoter activity in Saos-2 cells, whereas Sp1 used as a positive control activated the promoter. We also used the c-myc/ HO15.19 cell line to see whether c-Myc is directly involved in the LT-induced repression of the PDGF ß-receptor promoter activity (Fig. 5B). Transfection of LT did not repress, but almost doubled the activity of PDGF ß-receptor promoter. Sp1 strongly activated the promoter activity in this cell line. Furthermore, we examined the promoter activity in the Rb/ 3T3 cell line, which also failed to respond to LT. The expression level of PDGF ß-receptor was examined in this cell line at 0, 4, 8, 12, 24, 48 hours after serum stimulation following 48 hours' serum starvation and was compared with that of normal 3T3 cells. Expression of the receptor was higher in Rb/ cells, and no change was seen during the observed time period as judged by immunoblotting. c-Myc expression increased at 4 hours after serum stimulation even in the absence of pRb as reported elsewhere (Herrera et al., 1996), which was followed by a much slower decrease compared to the rapid decrease seen in normal 3T3 cells. This suggests that downregulation of the PDGF ß-receptor, as well as that of c-Myc, is impaired in the absence of pRb.
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p53 increases the PDGF ß-receptor promoter activity through NF-Y and Sp1 binding motifs
We showed that the p53-binding defective LT was unable to repress the PDGF ß-receptor promoter activity. We therefore tested whether p53 has any activity at all on the promoter. In a previous study, we showed that p73 bound NF-Y and repressed the promoter activity, but p53 did not (Hackzell et al., 2002
). Careful evaluation of the effect of p53 on various PDGF ß-receptor promoter constructs in 3T3 cells showed a small but significant increase in the activity (Fig. 6A). The 3'SacI/ApaI construct, consisting of a 142-bp sequence located downstream of the initiation site, did not respond to p53 co-transfection (data not shown). The HindIII/SacI construct containing the CCAAT motif showed a 50% increase of basic activity when p53 was co-transfected. The SacI/SacI constructs containing both the CCAAT- and Sp1-binding motifs showed a 70% increase of the activity by p53. However, the mutation in the CCAAT motif in the SacI/SacI still maintained 50% of the activation by p53 expression.
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These results indicate that the promoter activation by p53 is likely to be dependent on binding of both Sp1 and NF-Y. In order to analyse more exactly the effect of p53, we used the SL-2 cell line lacking both endogenous Sp1 and NF-Y (Courey and Tjian, 1998; Magana et al., 2000; Börestam et al., 2003). In this cell line, the SacI/SacI PDGF ß-promoter showed very low activity (Fig. 6B). Co-transfection of Sp1 alone yielded a stronger activation than NF-Y alone, and Sp1 and NF-Y together yielded a synergistic activation of the promoter. Addition of p53 yielded an additive effect on NF-Y transfection but a small synergism was observed with Sp1 on the promoter activation. All these factors together brought about a significant activation due to the synergisms between Sp1 and NF-Y as well as of p53 and Sp1. It is thus likely that p53 activates the promoter, chiefly through the Sp1-binding site.
SV40LT and p53 bind the proximal PDGF ß-receptor promoter in vivo
In order to see whether LT binds the PDGF ß-receptor promoter in vivo, Chip assays were performed. ST15A cells cultured at both permissive and non-permissive temperatures were used. PCR amplification of the proximal PDGF ß-receptor promoter was carried out with DNA extracted from the immunocomplex precipitated by an anti-SV40LT antibody or anti-p53 antibody. This promoter region contains the Sp1-binding sites, the CCAAT motif, and the initiation site. As a control, we included about 1.5 kbp of upstream sequence of the same promoter, lacking these consensus motifs. Fig. 7A shows that the PDGF ß-receptor promoter sequence was significantly enriched in the complex obtained with the anti-LT antibody in ST15A cells at 33°C when LT is expressed in the cells. No PDGF ß-receptor promoter sequence was detected in the immunocomplex obtained at 39°C or when the antibody was substituted with mouse IgG (Fig. 7B). When the p53-antibody was used for immunoprecipitation, the promoter sequence was amplified. Binding of p53 to the promoter was clearly stronger at 39°C than at 33°C (Fig. 7A,B). The input cell lysate as well as the precipitated DNA were also analysed at one-third dilutions. The amount of total protein in ST15A cells at different temperatures was examined by immunoblotting (Fig. 7C). As expected, LT was expressed only at 33°C, and p53 was expressed almost at the same level or slightly stronger at 33°C.
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In order to see whether LT binds the promoter in the absence of p53 and pRb, we made a Saos-2 cell clone stably expressing LT (Saos-2-LT), and used for Chip assays. In both parental and Saos-2-LT cell lines, the proximal promoter, but not the distal promoter, was precipitated by an NF-YB antibody used as a positive control (Fig. 7D,E). An anti-LT antibody bound the proximal promoter in Saos-2-LT cells, suggesting that LT can bind the promoter in the absence of pRb and p53. Expression of LT in Saos-2-LT and Saos-2 cells was compared by immunoblotting as shown in Fig. 7F.
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Discussion |
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It is possible that LT-bound pRb could act as a repressor by recruiting chromatin-remodelling complexes, such as histone deacetylases (HDACs) and the ATP-dependent SWI/SNF (Zhang et al., 2000). This is supported by our results that both of the LXCXE motif-mutated pRb-binding defective LTs markedly increased the promoter activity in NIH3T3 cells. In contrast, neither the p53-binding mutant nor the J domain mutant resulted in clear activation. The LXCXE motif and pRb create the most extensive interaction (Gjoerup et al., 2000
; Kim et al., 2001
). It should be noted that the activation on the PDGF ß-receptor promoter occurred through the CCAAT motif, the site needed for c-Myc- or p73-induced repression. Thus, the chromatin modification of this promoter area might be the key target of LT. However, the clear mechanism of the transactivation by the pRb-binding defective LT mutant remains to be elucidated.
Chip assays in ST15A cells with a p53 antibody revealed that p53 binds the promoter, and that the binding was stronger in the absence of LT at 39°C than at 33°C. Thus, LT may bind and sequester p53 from the promoter as the total amount of p53 in cells rather increased at 33°C, as judged by immunoblotting using total cell lysates including nuclear proteins. Downregulation of p53 mRNA expression at 39°C in this cell line was also reported previously (Hayes et al., 1991). It is notable that p53 binds the promoter, the bound p53 increases when LT is absent and the receptor expression is enhanced. In fact, the p53-binding area of LT overlaps with its DNA-binding surface (Li et al., 2003
). Although no clear p53 responsive element is found in this promoter area, LT seems to be capable of binding and repressing the promoter, partly through inhibiting the p53 action. Further characterisation of the LT and p53 binding sites on the promoter is underway in our laboratory.
Chip assays in the Saos-2 cells containing LT, confirmed that LT-binding of the promoter is not sufficient for the repression to occur. The presence of intact pRb and p53 is thus crucial. The proximal primer set was made so that the PCR product would contain the consensus sequences for Sp1 and NF-Y as well as the initiation site. In addition to binding to pRb and p53, resulting in the repression of the promoter, LT was also found to bind Sp1 (result not shown). It is possible that LT interferes with the activation of Sp1. As Sp1 interacts and activates NF-Y (Liang et al., 2001), LT might negatively affect the whole complex including both of the factors. In myc/ cells, co-transfection of Sp1 strongly activated the PDGF ß-receptor promoter (Fig. 5B). This suggests that Myc may actually repress the promoter activity not only by directly interacting with NF-Y, but also by sequestering Sp1 as reported (Gartel et al., 2001
). We could not show this activity of Myc in 3T3 cells, possibly because endogenous Myc was present (Molander et al., 2001
).
In agreement, the p53-binding defective LT is unable to repress the promoter activity. We previously found that levels of Myc and p73 increase following serum stimulation when they repress the promoter activity. So far of all cell-cycle regulating molecules investigated by us, only Np73 has been shown to activate the promoter (Hackzell et al., 2002
). The p53-mediated activation that we detected here is small yet significant and appears to involve the Sp1-binding site of the promoter. In fact, in combination with Sp1, the activation can be further enhanced. It has been reported that induction of p53 results in a complex formation with Sp1 through its C-terminus, dissociating HDAC1 from Sp1 on the p21 promoter (Lagger et al., 2003
). p53 would thus not only collaborate with Sp1 activation, but also prevent deacetylation of histones by releasing HDAC1 from Sp1. Whether it happens on this receptor promoter remains to be studied.
In conclusion, LT seems to severely affect the PDGF ß-receptor promoter by interfering with the activation by NF-Y and Sp1, through a mechanism involving Myc, pRb and p53. There is a strong concern as to whether SV40 is involved in the pathogenesis of human tumours. This possibility exists as several spontaneously occurring human tumours, such as mesothelioma, osteosarcoma and other brain tumours contain DNA sequences derived from the SV40 genome (Klein et al., 2002). However, defective suppressor genes in tumours may render them unresponsive to the repression by LT, thus tumours could maintain the growth factor receptor expression uncoupled from the cell cycle.
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Acknowledgments |
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Footnotes |
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Present address: IDEXX Scandinavia AB, Storrymningsvägen 5, SE-748 30 Österbybruk, Sweden
Present address: Department of Molecular Biology, University of Occupational and Environmental Health, School of Medicine, Kitakyushu, 807-8555, Japan
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