Mutant p53 expression leads to suppression of p53-target gene expression, which is relieved upon silencing of mutant p53 expression

<p><b>Copyright information:</b></p><p>Taken from "Cancer-derived p53 mutants suppress p53-target gene expression—potential mechanism for gain of function of mutant p53"</p><p></p><p>Nucleic Acids Research 2007;35(6):2093-2104.</p><p>Published online 7 Mar 2007</p><p>PMCID:PMC1874625.</p><p>© 2007 The Author(s)</p> () H1299-based isogeneic cell lines stably expressing the six hot-spot p53 mutants (i.e. 175, 245, 248, 249, 273 and 282) either in the proline (Pro) or arginine (Arg) codon 72 polymorphic form were analysed for steady-state p53-target gene expression levels, as indicated, by reverse-transcriptase PCR reaction. H1299-cells stably expressing the temperature-sensitive p53 mutants, which adopt a wild-type conformation at 32°C, were grown at 32°C and used as positive controls for p53-target gene activation (WT-Pro and Arg). Levels of were also evaluated. ( and ) H1299-cells expressing p53 mutant R273H in the proline (P) or arginine (R) codon 72 polymorphic form were transfected with either control scrambled siRNA or -specific siRNA or left untransfected (−). Cells were collected 48 h later for mRNA analysis of the indicated target genes by RT-PCR reaction (B) or by immunoblot analysis (C). () Vector-expressing H1299 cells were transfected with control scrambled siRNA, -specific siRNA, cDNA or left untransfected (−), and the levels of target genes were analysed by RT-PCR reaction

Mutant p53 expression results in down-regulation of p53-target gene promoters

<p><b>Copyright information:</b></p><p>Taken from "Cancer-derived p53 mutants suppress p53-target gene expression—potential mechanism for gain of function of mutant p53"</p><p></p><p>Nucleic Acids Research 2007;35(6):2093-2104.</p><p>Published online 7 Mar 2007</p><p>PMCID:PMC1874625.</p><p>© 2007 The Author(s)</p> () H1299 cells were transiently transfected with plasmids expressing the empty vector (pCDNA) (V), wild-type (WT) or the p53 mutant constructs either in a Proline (P) or Arginine (R) form, together with the various reporter plasmids expressing the firefly luciferase gene under the transcriptional control of the indicated gene promoters. Luciferase activity was analysed 24 h post-transfection. Transfections were carried out in triplicates and at least three independent times and the standard deviations are indicated. Wild-type p53 expression led to massive induction of the p53-target gene promoters, and hence, the graphs are depicted to highlight the down-regulation by mutant p53. () Representative western blot analysis was performed using half the samples from the transfections to determine the expression status of p53 in all cases. Actin levels show loading control

Presence of p53 binding sites on p21 promoter is not required for down-regulation by mutant p53

<p><b>Copyright information:</b></p><p>Taken from "Cancer-derived p53 mutants suppress p53-target gene expression—potential mechanism for gain of function of mutant p53"</p><p></p><p>Nucleic Acids Research 2007;35(6):2093-2104.</p><p>Published online 7 Mar 2007</p><p>PMCID:PMC1874625.</p><p>© 2007 The Author(s)</p> () Schematic of promoter indicating the position of the p53 binding sites (S1 and S2) as described (). () Luciferase reporter assays were preformed by transfecting vector, wild-type p53, or the indicated p53 mutant cDNA constructs into H1299 cells as described, with the various promoter constructs. Transfections were carried out in triplicates and at least three independent times and the standard deviations are indicated

Silencing of p53 expression results in reduced colony formation and elevated cell death

<p><b>Copyright information:</b></p><p>Taken from "Cancer-derived p53 mutants suppress p53-target gene expression—potential mechanism for gain of function of mutant p53"</p><p></p><p>Nucleic Acids Research 2007;35(6):2093-2104.</p><p>Published online 7 Mar 2007</p><p>PMCID:PMC1874625.</p><p>© 2007 The Author(s)</p> ( and ) p53 expression was silenced as described in HUH-7 and T47D cells using pSuper-based control or -specific siRNA, or were left untransfected. Cells were selected for two weeks on G418. Parallel cultures were used for semi-quantitative RT-PCR analysis of expression status (representative results shown for HUH-7 cells) (A) and colonies were visualized by staining with crystal violet solution (B, upper panel). All experiments were performed at least thrice independently and representative results are shown. Colonies were counted both manually and using colony count software. Similar results were obtained using both methods. Data are presented as the mean ± SD error of the mean. () Cell death was analysed by measuring the sub-G1 DNA content reflective of apoptotic cells, by flow cytometry. HUH-7 cells were transfected with scrambled or p53 siRNA (100 or 200 nM) and the percentage cell death was determined 5 days after transfection. Representative results are shown

Risk estimates (OR^* and 95% CI^#) for leukemia for individual SNP.

<p>*: OR – Odds ratio.</p>#<p>: CI - confidence interval.</p

Silencing of mutant p53 expression results in upregulation of p53-target gene expression

<p><b>Copyright information:</b></p><p>Taken from "Cancer-derived p53 mutants suppress p53-target gene expression—potential mechanism for gain of function of mutant p53"</p><p></p><p>Nucleic Acids Research 2007;35(6):2093-2104.</p><p>Published online 7 Mar 2007</p><p>PMCID:PMC1874625.</p><p>© 2007 The Author(s)</p> ( and ) Mutant p53 harbouring HUH-7 hepatoma cells (A220G) were untransfected (−) or transfected with either control scrambled siRNA or p53-specific siRNA. Cells were collected 48 h later for mRNA analysis of the indicated target genes by reverse-transcriptase (RT) PCR reaction (A) or by immunoblotting with the specific antibodies (B). () RT-PCR target gene analysis was similarly performed in T47D breast cancer cells (C194T) (left panel) and CNE-2 nasopharyngeal carcinoma cells (R280T) (right panel). () mRNA analysis shows direct comparison of p53-regulated gene expression as well as p53-independent and expression levels in all three cell lines

Treatment with Trichostatin A relieves mutant p53-mediated suppression of p53-target genes, which is reduced in cells lacking p300

<p><b>Copyright information:</b></p><p>Taken from "Cancer-derived p53 mutants suppress p53-target gene expression—potential mechanism for gain of function of mutant p53"</p><p></p><p>Nucleic Acids Research 2007;35(6):2093-2104.</p><p>Published online 7 Mar 2007</p><p>PMCID:PMC1874625.</p><p>© 2007 The Author(s)</p> ( and ) Luciferase reporter assays were performed as described above using the indicated p53 constructs in the presence (+) or absence (−) of trichostatin-A (TSA). Cells were pre-treated with TSA (100 ng/ml) for 6 h before transfection and TSA was present during transfection and luciferase activity was analysed 18 h after transfection. Promoter activity was determined using the and the promoter constructs ) The level of luciferase activity in mutant p53-transfected cells are shown as the percentage activity compared to the vector-transfected cells, which was set at 100%. () H1299-based isogenic cells lines stably expressing the indicated mutant p53 in either the proline (P) or arginine (R) codon 72 polymorphic form were treated in the presence (+) or absence (−) of TSA as described for 18 h and the levels of and mRNA were analysed by reverse-transcriptase PCR reaction. () p53-target gene promoter activity was determined using the or promoter constructs in p300 deficient (pancreatic cancer cell line PaCL4) or proficient (lung adenocarcinoma H1299) cells

Risk estimates (OR and 95% CI) for lung cancer for individual SNP.

<p>Risk estimates (OR and 95% CI) for lung cancer for individual SNP.</p

Four marker haplotype frequencies and association with lung cancer.

<p>Four marker haplotype frequencies and association with lung cancer.</p

Graph showing pairwise r² linkage disequilibrium (LD). Squares in red with no numerical indicates complete LD between markers.

<p>Graph showing pairwise r² linkage disequilibrium (LD). Squares in red with no numerical indicates complete LD between markers.</p