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Item Low Levels of p53 Protein and Chromatin Silencing of p53 Target Genes Repress Apoptosis in Drosophila Endocycling Cells(2014-09) Zhang, Bingqing; Mehrotra, Sonam; Ng, Wei Lun; Calvi, Brian RApoptotic cell death is an important response to genotoxic stress that prevents oncogenesis. It is known that tissues can differ in their apoptotic response, but molecular mechanisms are little understood. Here, we show that Drosophila polyploid endocycling cells (G/S cycle) repress the apoptotic response to DNA damage through at least two mechanisms. First, the expression of all the Drosophila p53 protein isoforms is strongly repressed at a post-transcriptional step. Second, p53-regulated pro-apoptotic genes are epigenetically silenced in endocycling cells, preventing activation of a paused RNA Pol II by p53-dependent or p53-independent pathways. Over-expression of the p53A isoform did not activate this paused RNA Pol II complex in endocycling cells, but over-expression of the p53B isoform with a longer transactivation domain did, suggesting that dampened p53B protein levels are crucial for apoptotic repression. We also find that the p53A protein isoform is ubiquitinated and degraded by the proteasome in endocycling cells. In mitotic cycling cells, p53A was the only isoform expressed to detectable levels, and its mRNA and protein levels increased after irradiation, but there was no evidence for an increase in protein stability. However, our data suggest that p53A protein stability is regulated in unirradiated cells, which likely ensures that apoptosis does not occur in the absence of stress. Without irradiation, both p53A protein and a paused RNA pol II were pre-bound to the promoters of pro-apoptotic genes, preparing mitotic cycling cells for a rapid apoptotic response to genotoxic stress. Together, our results define molecular mechanisms by which different cells in development modulate their apoptotic response, with broader significance for the survival of normal and cancer polyploid cells in mammals.Item Modeling cancer predisposition: Profiling Li-Fraumeni syndrome patient-derived cell lines using bioinformatics and three-dimensional culture models(2015-10-07) Phatak, Amruta Rajendra; Herbert, Brittney-Shea; Liu, Yunlong; Mendonca, Marc S.; Wells, Clark D.Although rare, classification of over 200 hereditary cancer susceptibility syndromes accounting for ~5-10% of cancer incidence has enabled the discovery and understanding of cancer predisposition genes that are also frequently mutated in sporadic cancers. The need to prevent or delay invasive cancer can partly be addressed by characterization of cells derived from healthy individuals predisposed to cancer due to inherited "single-hits" in genes in order to develop patient-derived samples as preclinical models for mechanistic in vitro studies. Here, we present microarray-based transcriptome profiling of Li-Fraumeni syndrome (LFS) patient-derived unaffected breast epithelial cells and their phenotypic characterization as in vitro three-dimensional (3D) models to test pharmacological agents. In this study, the epithelial cells derived from the unaffected breast tissue of a LFS patient were cultured and progressed from non-neoplastic to a malignant stage by successive immortalization and transformation steps followed by growth in athymic mice. These cell lines exhibited distinct transcriptomic profiles and were readily distinguishable based upon their gene expression patterns, growth characteristics in monolayer and in vitro 3D cultures. Transcriptional changes in the epithelial-to-mesenchymal transition gene signature contributed to the unique phenotypes observed in 3D culture for each cell line of the progression series; the fully transformed LFS cells exhibited invasive processes in 3D culture with disorganized morphologies due to cell-cell miscommunication, as seen in breast cancer. Bioinformatics analysis of the deregulated genes and pathways showed inherent differences between these cell lines and targets for pharmacological agents. After treatment with small molecule APR-246 that restores normal function to mutant p53, we observed that the neoplastic LFS cells had reduced malignant invasive structure formation from 73% to 9%, as well as an observance of an increase in formation of well-organized structures in 3D culture (from 27% to 91%) by stereomicroscopy and confocal microscopy. Therefore, the use of well-characterized and physiologically relevant preclinical models in conjunction with transcriptomic profiling of high-risk patient derived samples as a renewable laboratory resource can potentially guide the development of safer and more effective chemopreventive approaches.