Project description:Investigation of microRNA expression level changes in human colon cancer cell lines in response to p53 activation by Inauhzin (INZ), and identification of novel p53 target microRNAs. HCT116 p53+/+ and HCT116 p53-/- cells were treated with DMSO or 4μM INZ and harvested at 18 hours post treatment. Cells were then homogenized in TriZol reagents. Duplicates were included for each sample. Total RNA was extracted by following the manufacturerâs standard instructions (Invitrogen).
Project description:Investigation of microRNA expression level changes in human colon cancer cell lines in response to p53 activation by Inauhzin (INZ), and identification of novel p53 target microRNAs.
Project description:In most lymphomas, p53 signaling pathway is inactivated by various mechanisms independent to p53 gene mutations or deletions. In many cases, p53 function is largely regulated by alterations in the protein abundance levels by the action of E3 ubiquitin-protein ligase MDM2, targeting p53 to proteasome-mediated degradation. In the present study, an integrating transcriptomics and pro-teomics analysis was employed to investigate the effect of p53 activation by a small-molecule MDM2-antagonist, nutlin 3a, on three lymphoma cell models following p53 activation. Our analy-sis revealed a system-wide nutlin 3a-associated effect in all examined lymphoma types, identifying in total of 4037 differentially affected proteins involved in a plethora of pathways, with significant heterogeneity among lymphomas. Our findings include known p53-targets and novel p53 activa-tion effects, involving transcription, translation, or degradation of protein components of path-ways, such as a decrease in key members of PI3K/mTOR pathway, heat-shock response, and gly-colysis, and an increase in key members of oxidative phoshosphorylation, autophagy and mito-chondrial translation. Combined inhibition of HSP90 or PI3K/mTOR pathway with nutlin 3a-mediated p53-activation enhanced the apoptotic effects suggesting a promising strategy against human lymphomas. Integrated omic profiling after p53 activation offered novel insights on the regulatory role specific proteins and pathways may have in lymphomagenesis.
Project description:Signaling trough cytoplasmic or nuclear action of p53 is a major response mechanism to cellular stresses. Here we perform transcriptomics after p53 re-expression on a CRISPR/Cas9 knock out background to reveal a distinct starvation-specific transcriptome response and novel nutrient-dependent p53 target genes.
Project description:Several genome-wide transcriptome analyses that focused on p53-induced cellular responses in many cellular contexts have continued to expand the already vast p53-regulated transcriptional networks. To investigate post-transcriptional controls as an additional dimension of p53-directed gene expression responses we performed translatome analysis by polysomal profiling on MCF7 cells treated with Doxorubicin and Nutlin-3a. A comparison between the transcriptome and the translatome revealed a large number of uncoupled genes, whose transcription changes did not correlate with translation changes. Overall, we establish p53 as a master regulator of translational control and identify many p53 target genes affecting translation that can contribute to p53-dependent cellular responses. Keywords: p53, transcriptome, translatome, polysomal RNA, subpolysomal RNA, uncoupling, Doxorubicin, Nutlin-3a Total RNA (tot) was extracted from MCF7 vector cells after 16h of treatment with Doxorubicin (1.5uM) and Nutlin-3a (10uM) or DMSO (solvent, as control treatment). Polysomal profiling was performed after the same conditions. We collected all subpolysomal mRNA fractions (sub) and the polysomal ones (pol) after sucrose gradient fractionation of cytoplasmic lysates to analyze separately mRNAs that are not actively translated from those that are considered in active translation, respectively. Experiments were performed in three biological replicates.
Project description:Several genome-wide transcriptome analyses that focused on p53-induced cellular responses in many cellular contexts have continued to expand the already vast p53-regulated transcriptional networks. To investigate post-transcriptional controls as an additional dimension of p53-directed gene expression responses we performed translatome analysis by polysomal profiling on MCF7 cells treated with Doxorubicin and Nutlin-3a. A comparison between the transcriptome and the translatome revealed a large number of uncoupled genes, whose transcription changes did not correlate with translation changes. Overall, we establish p53 as a master regulator of translational control and identify many p53 target genes affecting translation that can contribute to p53-dependent cellular responses. Keywords: p53, transcriptome, translatome, polysomal RNA, subpolysomal RNA, uncoupling, Doxorubicin, Nutlin-3a
Project description:Ceramides are important participants of signal transduction, regulating fundamental cellular processes. Here we report the mechanism for activation of p53 tumor suppressor by C16-ceramide. C16-ceramide tightly binds within the p53 DNA binding domain (Kd ~ 60 nM), in close vicinity to the Box V motif. This interaction is highly selective towards the ceramide acyl chain length with its C10 atom being proximal to Ser240 and Ser241. Ceramide binding stabilizes p53 and disrupts its complex with E3 ligase MDM2 leading to the p53 accumulation, nuclear translocation and activation of the downstream targets. This is a novel physiological mechanism of p53 activation, which is fundamentally different from the canonical p53 regulation through protein-protein interactions or post-translational modifications. The discovered mechanism is triggered by serum or folate deprivation implicating it in the cellular response to nutrient/metabolic stress. Our study establishes C16-ceramide as the first natural small molecule activating p53 through the direct binding.
