Figure 1.
A. p53 knockdown in MCF-7 cells. A stable MCF-7 subline was established by transfection pGeneSupressor/p53siRNA followed by G418 selection. Control and MCF-7/p53siRNA cells were treated with doxorubicin (Dox) at indicated concentrations for 20 h. Protein levels of p53, p21 and actin were detected by Western blot. B & C. Upregulation of p73 β in MCF-7 (B) and HCT-116 (C) cells with p53 knockdown/knockout. Protein levels of p53, p73 β and Actin in control and p53 knockdown/knockout cells were detected with Western blot. D. Regulation of p73 by p53 in transient transfection system. MCF-7 cells were transiently transfected with control vector, pGeneSupressor/p53siRNA, pCMV/mtp53 (135G/A) and pCMV/wtp53, respectively. Protein levels of p53, p73 β and Actin were detected using Western blot.
Figure 2.
p53 inactivation mediated upregulation of p73 is primarily regulated at the transcriptional level.
A & B. p53 inactivation upregulates TAp73 but not DNp73 mRNA levels. Relative mRNA levels of TAp73 (A) and DNp73 (B) in control cells (with wtp53) and p53 knockdown/knockout (p53KD/KO) MCF-7 and HCT-116 cells were detected with real-time RT-PCR using TAp73 and DNp73 specific primers. Specific mRNA levels were normalized with β-Actin mRNA. C & D. Loss of p53 induces the activation of p73 promoter in stable MCF-7 and HCT-116 sublines. Cells of each subline were co-transfected with luciferase reporter construct encoding full length (−2713 to +77) p73 promoter (p73-PF), pSV-β-gal. Luciferase activity was measured 40 hours after transfection. The experiments were performed at least three times in triplicates. E. Transfaction of p53 siRNA and mutant p53 induces p73 promoter activity in MCF-7 cells: MCF-7 cells were co-transfected with reporter construct encoding full length p73 promoter (p73-PF), pSV-β-gal, and vectors of control siRNA, p53siRNA, wild type p53 (wtp53) or mutant p53 (mtp53). Luciferase activity was determined as described above. (* p<0.01).
Figure 3.
p53 inactivation mediated upregulation of p73 is independent of p53's binding to the p73 promoter.
A. p53 binds to the p73 promoter. EMSA was performed using 32P-labeled 61 bp oligonucleotide (−2634 to −2574) containing putative p53-binding sequence in the p73 promoter and nuclear extracts of MCF-7 control and MCF-7/p53siRNA cells. Control and p53 knockout (p53−/−) HCT116 cells were used as control. Arrow indicates p53 bound to the p73 promoter sequence. B. Deletion of p53 binding site in the p73 promoter did not abrogate p53-knockdown mediated upregulation of p73 promoter activity. MCF-7 cells were transfected with the luciferase reporter constructs encoding full length p73 promoter, p73PF (PF) or the promoter lacking 61 bp p53 binding sequence, p73-PFΔ61 (D61) in the presence or absence of p53 knockdown with siRNA.
Figure 4.
Mapping of the DNA sequence that is responsible for p53 inactivation mediated p73 upregulation.
A. Reporter constructs of TAp73 promoter with a serious of deletions from the 5′ end. Putative binding sites for E2F and p53 are depicted. The drawing is not proportional to the actual size. B. MCF-7 cells were co-transfected with luciferase reporter constructs with different lengths of p73 promoter, pSV-β-Gal, and vectors of control siRNA or p53siRNA. Luciferase activity was measured 40 hours after transfection, which was followed by β-galactosidase normalization.
Figure 5.
Critical role E2F-1 in p53 knockdown-mediated upregulation of p73 transcription.
A. Additive effect of E2F-1 overexpression on p53 knockdown mediated increase in p73 promoter activity. MCF-7 cells were co-transfected with p73-PF/luciferase, pSV-β-Gal, and control or p53siRNA plasmids either in presence or in absence of E2F-1. Luciferase and β-galactosidase activity was determined 40 hours post-transfection. B. TAp73 mRNA levels in MCF-7 cells transfected with control vector, p53siRNA and/or E2F-1 cDNA. MCF-7 cells were transiently transfected with either control siRNA or p53-specific siRNA either in the presence or in the absence of E2F-1. RT-PCR analysis was performed using gene-specific primers. C & D. E2F-1 binding is required for p53 knockdown mediated increase in p73 promoter activity. MCF-7 cells were co-transfected with control or p53siRNA vector and reporter construct encoding wild type or mutant p73 promoter (p73PVUII, −220 to +71), in addition to pSV-β-Gal plasmid. The mutant PVUII promoter fragment contains mutant E2F-1 binding sites at −155 and −132 (C). Luciferase and β-gal activity was determined 40 hours post- transfection (D). E. Occupancy of the E2F responsive element in the TAp73 promoter by E2F-1 is enhanced in MCF-7/p53siRNA cells. Detected with chromatin immunoprecipitation (ChIP) assay, DNA fragment of the TAp73 promoter was amplified from the complexes immunoprecipitated with E2F-1 antibody from the paired cell lines. Input row were the DNA fragment amplified from the extracts before immunoprecipitation. In the control immunoglobulin G (IgG) reaction, PCR was done in the eluates from beads collected after preclearing of these extracts with normal rabbit serum.
Figure 6.
p21 is a mediator of p53 inactivation induced upregulation of p73 transcription.
A. Overexpression of p21 abolishes p73 upregulation in control and MCF-7/p53siRNA cells. MCF-7/control and MCF-7/p53siRNA cells were cotransfected with p73-PF/pSV-β-Gal and control vector or pcDNA3/p21. Cell lysate was collected for luciferase assay 40 hours post-transfection. All the experiments were performed at least three times in triplicates. B. Overexpression of wtp53 reverses p53siRNA induced p73 transcription in the presence or absence of E2F-1 overexpression. p73-PF/pSV-β-Gal and pcDNA3/E2F-1 or control vector were cotransfected with the plasmids encoding control siRNA, p53siRNA or wtp53 into MCF-7 cells. Luciferase activity was determined as described above. C. Overexpression of p21 abrogates p53siRNA induced p73 transcription in the presence or absence of E2F-1 overexpression. p73-PF/pSV-β-Gal and pcDNA3/E2F-1 or control vector were cotransfected with the plasmids encoding control siRNA, p53siRNA or pcDNA3/p21 into MCF-7 cells. Luciferase activity was determined as described above.