Project description:The human TP53 gene is frequently mutated in tumors and cell lines. Unlike other tumor suppressors that are commonly inactivated by deletions or nonsense mutations, the majority of p53-mutations are missense point mutations that result in the expression of a full-length protein with an altered amino acid that has lost sequence specific DNA-binding. Expression of mutant p53 (mutp53) confers advantages to tumor cells and transcriptional regulation of several genes mediating the beneficial effects has been shown to play a role. However, molecular mechanisms of transcriptional regulation by mutp53 are still poorly understood. We used the glioblastoma-derived U-251 MG human cell line endogenously expressing mutp53 protein (R273H mutation) to analyze gene expression profiles on Agilent Whole Human Genome Microarray after transient and stable depletion of mutp53 expression. Gene expression data was correlated with a ChIP study on a custom tiling array to understand the contribution of endogenously expressed mutp53 to transcriptional regulation. This series of microarray experiments contains the gene expression profiles of glioblastoma-derived U-251 MG human cell lines engineered to constitutively express a p53-specific shRNA or scrambled control shRNA. To reverse the effect of mutp53 depletion, stable clones were modified by stable integration of a mutp53-R273H expression construct or empty pCDNA3 vector as a control. In addition, we performed gene expression analysis of U-251 MG cells transiently transfected with p53-specific siRNA or control siRNA (3 biological replicates each).
Project description:The completion of the Plasmodium falciparum clone 3D7 genome provides a basis on which to conduct comparative proteomics studies of this human pathogen. Here, we applied a high-throughput proteomics approach to identify new potential drug and vaccine targets and to better understand the biology of this complex protozoan parasite. We characterized four stages of the parasite life cycle (sporozoites, merozoites, trophozoites and gametocytes) by multidimensional protein identification technology. Functional profiling of over 2,400 proteins agreed with the physiology of each stage. Unexpectedly, the antigenically variant proteins of var and rif genes, defined as molecules on the surface of infected erythrocytes, were also largely expressed in sporozoites. The detection of chromosomal clusters encoding co-expressed proteins suggested a potential mechanism for controlling gene expression. Keywords: ordered
Project description:p53 is a pivotal tumor suppressor and a major barrier against cancer. We now report that silencing of the Hippo pathway tumor suppressors LATS1 and LATS2 in non-transformed mammary epithelial cells reduces p53 phosphorylation and increases its association with the p52 NF-κB subunit. Moreover, it partly shifts p53’s conformation and transcriptional output towards a state resembling cancer-associated p53 mutants, and endow p53 with the ability to promote cell migration. Notably, LATS1 and LATS2 are frequently downregulated in breast cancer; we propose that such downregulation might benefit cancer by converting p53 from a tumor suppressor into a tumor facilitator.
Project description:Mutant p53 proteins, resulting form frequent TP53 tumor suppressor missense mutations, possess gain-of-function activities and are among the most widespread and robust oncoproteins in human tumors. They are potentially important but understudied therapeutic targets. No studies to date have distinguished common, therapeutically relevant mutant p53 gain-of-function effects, from effects specific to different mutant variants and cell backgrounds. Here we identify 26S proteasome machinery as the common downstream effector controlled by mutant p53s in Triple Negative Breast Cancer (TNBC - aggressive carcinomas with TP53 as the most frequently mutated locus) and conserved in other human cancers. We have identified this pathway using a combination of single-model, multi-method vertical analysis (whole cell proteome, RNA sequencing an ChIP sequencing) and multi-cell line, horizontal analysis of transcriptiomes. We found that different missense mutant p53s regardless of the cell background transcriptionaly activate whole 26S proteasome machinery. Proteasome activity is significantly increased in p53 mutant versus wild-type or knockdown/null status - in cellular and mouse models as well as in human breast tumors. Increased proteasome activity leads to inhibition of tumor suppressive pathways. The control of mutant p53 over proteasome transcription and activity results in the increased resistance to proteasome inhibitors. By combining the mutant p53 targeting agents and proteasome inhibitor we were able to overcome the “bounce-back” proteasome inhibitor resistance mechanism in mutant p53 bearing TNBC cells and xenografts in vivo.
Project description:To evaluate the effect on gene expression by shRNA-58335, we evaluated gene expression by microarray analysis. Gene expression was measured in Huh-7 cells stably expressing shRNA-58335 or control shRNA after infection with adenovirus expressing p53 (Ad-p53) or LacZ (Ad-LacZ).
Project description:To evaluate the effect on gene expression by lincRNAs, we knocked down three lincRNAs and evaluated the gene expression by microarray analysis. Gene expression was measured in Hep3B cells transfected with siRNAs targeting the indicated lincRNAs or negative control siRNA and infected with adenovirus expressing p53 (Ad-p53) or LacZ (Ad-LacZ).
Project description:HCT116 and HCT116 p53 null were exposed to two Aurora kinase inhibitors (CYC116 and ZM447439) at cytotoxic concentrations. Within 4-5 weeks we were able to select and isolate 10 drug resistant clones from each group. 3 clones were selected for gene expression profiling using Affymetrix microarrays (Human Gene 1.0 ST Array). The samples in each group were initially given the code names as follows. Group 1. [R1.3, R4.2, R6.3: CYC116 p53 wild type], Group 2. [R8.7, R9.7, R10.7:CYC116 p53 null], Group 3. [R7.1, R15.1, R16.1:ZM447439 p53 wild type], and Group 4. [R1.5, R3.5, R4.5:ZM447439 p53 null). For publication purpose the names were later changed to, Group 1. [R1.1, R1.2, R1.3: CYC116 p53 wild type], Group 2. [R2.1, R2.2, R2.3:CYC116 p53 null], Group 3. [R3.1, R3.2, R3.3:ZM447439 p53 wild type], and Group 4. [R4.1, R4.2, R4.3:ZM447439 p53 null].
Project description:This experiment was performed to determine which gene promoters mutant p53 binds and transcriptionally regulates in order to understand how mutant p53 accomplishes its gain of function phenotype.
Project description:To investigate the impact of combined Rb and p53 loss in mammary tumorigenesis, we used transgenic and viral approaches to delete Rb and p53 floxed alleles specifically in the mouse mammary epithelium. Although MMTV-Cre (NLST) targets stem/bi-potent progenitors in the mammary gland, a subset of MMTV-Cre:Rbf/f;p53f/f mice developed non-mammary tumors. Thus, freshly isolated primary mammary epithelial cells from these animals were transplanted into the mammary fat pads of immunodeficient mice and monitored for tumor formation. In addition, primary MECs were isolated from Cre-negative Rbf/f;p53f/f mice, infected with Ad-Cre followed by orthotopic transplantation. In all these cases, resulting tumors shared similar spindle-shape histology, expressed high levels of vimentin, a mesenchymal marker, but not E-cadherin, a luminal marker, and were classified as adeno-sacrcomatoid/spindle-cell/mesenchymal-like breast cancer. We used aCGH to detect copy number alterations associated with Rb/p53 deletion. Tumor DNAs from MMTV-Cre: Rbf/f;p53f/f and Ad-Cre: Rbf/f;p53f/f conditional mutant mice are being compared to pooled tail DNAs in order to identify common alterations associated with Rb/p53 deficient tumorigenesis
Project description:Performing Chromatin IP of Klf2, Klf4, Klf5 and p53 in mouse embryonic stem cells with NimbleGen custom genomic tiling arrays, we sought to decipher Klf2, Klf4, Klf5-regulated genes. 12 samples: Chromatin IP of Klf2, Klf4, Klf5 and p53 in mouse embryonic stem cells with NimbleGen custom genomic tiling arrays; three independent experimental replicates for each experimental condition were performed.