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First published online 20 July 2004
doi: 10.1242/jcs.01228

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pRb, Myc and p53 are critically involved in SV40 large T antigen repression of PDGF ß-receptor transcription

Hidetaka Uramoto, Anders Hackzell^*, Daniel Wetterskog^*, Andrea Ballági, Hiroto Izumi and Keiko Funa^¶

Department of Cell Biology, Institute of Anatomy and Cell Biology, Göteborg University, Box 420, SE-405 30 Gothenburg, Sweden

View larger version (40K): [in a new window]   Fig. 1. (A) Downregulation of PDGF ß-receptor by LT on ST15A cells, containing a temperature-sensitive mutant of LT cultured at 33°C or 39°C. The amount of PDGF receptor binding was analysed by addition of serial dilutions of cold PDGF ligand to compete with [125I]PDGF ligand for cell binding. The -receptor was depleted by preincubation of cells with PDGF-AA at 37°C. (B) Photomicrographs of ST15A cells immunostained with a PDGF ß-receptor antibody after culture at 33°C (left) and after differentiation at 39°C (right).

View larger version (29K): [in a new window]   Fig. 2. (A) Immunoblotting with PDGF ß-receptor in 3T3 fibroblasts transiently transfected with LT, small t (st) or expression vector using Fugene cultured with 10% FCS. Lane 1: non-transfected cells, lane 2: 2 µg pcDNA3 vector, lane 3: 1 µg LT and 1 µg pcDNA3 vector, lane 4: 1 µg st and 1 µg pcDNA3 vector. (B) No alteration of PDGF ß-receptor expression in myc-null HO15.19 cells was shown by immunoblotting after transfection with LT, or pcDNA3 vector. Immunoblots with anti-LT and anti-actin antibodies are shown. (C) Expression of PDGF ß-receptor mRNA at 0, 2, 4 and 8 hours after transfection of LT or pcDNA3 vector in 3T3 cells. The RT-PCR products for PDGF ß-receptor, and actin were run on an agarose gel and are also shown as relative ratios of PDGF ß-receptor to actin obtained by densitometric analysis.

View larger version (21K): [in a new window]   Fig. 3. Effects of PDGF ß-receptor promoter activity by LT in 3T3 cells. (A) Schematic illustration of the promoter with relevant restriction enzyme sites. The GC-rich area (white box) and CCAAT motif (black box) are shown. (B) Relative luciferase activity of the SacI/SacI-reporter construct containing CCAAT motif co-transfected with st, LT, and vector alone (Mock). Basic vector without promoter was used as a basal control. Values represent mean promoter activity, and error bars indicate standard deviation of triplicate samples. A representative result of three repeated experiments is shown. The expression levels of LT and st are shown by immunoblotting after transfection. (C) The HindIII/SacI-reporter construct containing CCAAT motif, the MluI/SacI-reporter construct containing CCAAT motif and GC box or the SacI/SacI-construct was co-transfected with Sp1, LT or expression vector alone (mock). The expression of Sp1 is shown by immunoblotting. (D) The SacI/SacI construct was co-transfected with Sp1, LT or vector in the presence of DNNFYA and compared with the effect of LT in the absence of DNNFYA. The expression of NF-YA and DNNFYA is shown by immunoblotting.

View larger version (24K): [in a new window]   Fig. 4. Binding to both p53 and pRb is necessary for LT repression on PDGF ß-promoter activity and the LXCXE Rb-binding mutants enhance the promoter activity via CCAAT motif. (A) 3T3 cells were co-transfected with the SacI/SacI construct together with H42Q, 434-444, C105G, LTK1 or vector. The expression of all the LT vectors used in the assays is shown by immunoblotting. (B) 3T3 cells were co-transfected with the HindIII/SacI, SacI/SacI or basic reporter construct together with LT, K1, or vector alone. (C) 3T3 cells were co-transfected with the SacI/SacI construct containing wild type or mutated CCAAT motif together with vector alone, LTK1, DNNFYA or DNNFYA and LTK1. Luciferase activity was measured as described above.

View larger version (17K): [in a new window]   Fig. 5. LT is unable to repress PDGF ß-receptor promoter activity in (A) Saos-2 cells, (B) HO15.19 cells and (C) Rb null cells. Transfection of Sp1 was used as a positive control. Cells were co-transfected with the SacI/SacI construct together with LT or vector alone examined in 2 different vectors. Expression of LT is shown by immunoblotting. Luciferase activity was measured as described above. (D) Rb null cells express a stable level of PDGF ß-receptor during a cell cycle. Cells were harvested at indicated time points after serum stimulation of starved cells and used for immunoblots with antibodies against PDGF ß-receptor, c-Myc and actin. The lowest panel of 3T3 cells shows non-specific bands.

View larger version (26K): [in a new window]   Fig. 6. p53 increases PDGF ß-receptor promoter activity. (A) 3T3 cells were co-transfected with the HindIII/SacI or the SacI/SacI constructs (wild type or CCAAT-mutant) together with p53 or vector alone. The expression of p53 is shown by immunoblotting. (B) SL2 cells were co-transfected with the SacI/SacI construct and NF-Y, Sp1, NF-Y + Sp1 or vector alone in combination with p53. Luciferase activity was measured as described above.

View larger version (22K): [in a new window]   Fig. 7. LT and p53 bind PDGF ß-receptor promoter in vivo in ST15A cells and Saos-2 cells. (A,B) Chromatin was immunoprecipitated from ST15A cells cultured with anti-LT antibody, anti-p53 antibody or mouse IgG at 33°C (A) and 39°C (B). Immunoprecipitated DNA was purified and analysed by PCR together with whole cell lysate using primers specific for the proximal and distal areas of the PDGF ß-receptor promoter. (C) Total protein was extracted from ST15A cells cultured under both conditions and immunoblotted with anti-LT, anti-p53, and anti-actin antibodies. (D,E) Chromatin was immunoprecipitated from Saos-2 cells (D), and Saos-2-LT cells (E) stably expressing LT with normal rabbit serum, mouse IgG, anti-NF-YB, and anti-LT antibodies described as above. (F) Expression of LT and actin is shown in Saos-2 and Saos-2-LT cells by immunoblotting.