Critical role <i>E2F-1</i> in <i>p53</i> knockdown-mediated upregulation of <i>p73</i> transcription.

<p>A. Additive effect of <i>E2F-1</i> overexpression on <i>p53</i> knockdown mediated increase in <i>p73</i> 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 <i>E2F-1</i>. Luciferase and β-galactosidase activity was determined 40 hours post-transfection. B. <i>TAp73</i> mRNA levels in MCF-7 cells transfected with control vector, p53siRNA and/or <i>E2F-1</i> cDNA. MCF-7 cells were transiently transfected with either control siRNA or <i>p53</i>-specific siRNA either in the presence or in the absence of <i>E2F-1</i>. RT-PCR analysis was performed using gene-specific primers. C & D. <i>E2F-1</i> binding is required for <i>p53</i> knockdown mediated increase in <i>p73</i> promoter activity. MCF-7 cells were co-transfected with control or p53siRNA vector and reporter construct encoding wild type or mutant <i>p73</i> promoter (p73PVUII, −220 to +71), in addition to pSV-β-Gal plasmid. The mutant PVUII promoter fragment contains mutant <i>E2F-1</i> 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.</p

<i>p53</i> inactivation mediated upregulation of <i>p73</i> is independent of <i>p53</i>'s binding to the <i>p73</i> promoter.

<p>A. <i>p53</i> binds to the <i>p73</i> promoter. EMSA was performed using 32P-labeled 61 bp oligonucleotide (−2634 to −2574) containing putative <i>p53</i>-binding sequence in the <i>p73</i> promoter and nuclear extracts of MCF-7 control and MCF-7/p53siRNA cells. Control and <i>p53</i> knockout (p53−/−) HCT116 cells were used as control. Arrow indicates <i>p53</i> bound to the <i>p73</i> promoter sequence. B. Deletion of <i>p53</i> binding site in the <i>p73</i> promoter did not abrogate <i>p53</i>-knockdown mediated upregulation of <i>p73</i> promoter activity. MCF-7 cells were transfected with the luciferase reporter constructs encoding full length <i>p73</i> promoter, p73PF (PF) or the promoter lacking 61 bp <i>p53</i> binding sequence, p73-PFΔ61 (D61) in the presence or absence of <i>p53</i> knockdown with siRNA.</p

Mapping of the DNA sequence that is responsible for <i>p53</i> inactivation mediated <i>p73</i> upregulation.

<p>A. Reporter constructs of <i>TAp73</i> promoter with a serious of deletions from the 5′ end. Putative binding sites for <i>E2F</i> and <i>p53</i> 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 <i>p73</i> 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.</p

<i>p21</i> is a mediator of <i>p53</i> inactivation induced upregulation of <i>p73</i> transcription.

<p>A. Overexpression of <i>p21</i> abolishes <i>p73</i> 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 <i>p73</i> transcription in the presence or absence of <i>E2F-1</i> 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 <i>p21</i> abrogates p53siRNA induced <i>p73</i> transcription in the presence or absence of <i>E2F-1</i> 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.</p

Figure 1

<p>A. <i>p53</i> 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 <i>p53</i>, <i>p21</i> and actin were detected by Western blot. B & C. Upregulation of <i>p73</i> β in MCF-7 (B) and HCT-116 (C) cells with <i>p53</i> knockdown/knockout. Protein levels of <i>p53</i>, <i>p73</i> β and Actin in control and <i>p53</i> knockdown/knockout cells were detected with Western blot. D. Regulation of <i>p73</i> by <i>p53</i> 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 <i>p53</i>, <i>p73</i> β and Actin were detected using Western blot.</p