Browsing by Subject "Tumor Suppressor Protein p53"
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Item B-Catenin and K-ras Synergize to Form Wilm's Tumor with Concurrent p53 Pathways Modulation(2014-02-04) Hembd, Austin; Clark, Peter; DeGraff, DavidHumans can develop pediatric kidney tumors called Wilm's tumors. If one identifies the specific genes that cause Wilm's tumor, or that concomitantly change expression levels in the tumor tissue, then diagnosis and eventually drug targets for therapy are expedited. Characterizing genetic determinants in the mouse model can help actualize these future therapies. When the genes Kras and βCatenin are overexpressed in a mouse, it develops a renal tumor histologically identical to a human Wilm's tumor. Microarray analysis on mouse tumor tissue showed modulated expression levels of gene targets in the p53 tumor suppressor pathway. Immunohistochemistry stained mice tissue specifically for p53. In tissue with Kras and βcatenin overexpression, p53 staining is positive surrounding the tumor. RT qPCR measured levels of gene expression of p53 pathway associated genes. Combination mutants βCatenin and Kras were compared with controls. This PCR array analysis identified genes, such as cJun, Traf1, and Dapk1, that had significant expression changes in the combination mutant when compared to either mutant individually. The expression is modulated in a nonadditive fashion in Kras + βcatenin mutant tissues, which can explain the phenotype of Wilm's tumor in only double mutant mice. These genes individually represent targets for therapy in the future, and together represent an identifying fingerprint for diagnosis and prediction.Item Generation of a Novel D. melanogaster Platform to Elucidate Oncogenic Activity of Common Human p53 Missense Mutants(2014-02-04) Jakubowski, Brandon; D'Brot, Alejandro; Abrams, JohnThe tumor suppressor p53 prevents uncontrolled cell growth by three separate mechanisms: inducing apoptosis, initiating cell-cycle arrest, and activating DNA repair mechanisms in response to cell damage. Due to its central role in tumor eradication, it is unsurprising that p53 mutations are found in over half of human cancers. Unlike all other tumor suppressors however, 75% of these are missense mutations, with just six of them accounting for a third of all mutations found in the DNA binding domain of p53. Recent findings indicate that mutations in these "hotspot" locations may encode gain of function oncogenic activity to p53. Given their high prevalence, these mutations suggest a previously underappreciated selective advantage. We sought to decode this novel oncogenic activity of human p53 mutations by exploiting the Drosophila model system. This organism shares a similar p53 regulatory network with humans, as well as many of the same DNA repair and pro-apoptotic target genes. We recently showed that human p53, despite millions of years of evolutionary distance, complements loss of function mutations in the native fly p53 gene. We used six humanized p53 Drosophila strains previously generated in the lab; these contain a human p53 gene insertion, each with one of the six most commonly found missense mutations in patients. To study these mutations, we first profiled the expression patterns of wild type and mutant hp53 in the fly and their ability to rescue dp53 function. Expression levels of p53 were determined by immunofluorescence, while biological function was determined by the use of a GFP biosensor that specifically reports dp53 activity and acridine orange staining to identify dying cells in irradiated embryos. Expression studies demonstrate that the reporter is activated within stem cells in region 1 of the germarium, while its activation was absent in p53 null mutants. This phenotype was recovered with a dp53 insertion rescue. Additionally, two separately generated hp53+ strains show unusually elevated levels of expression compared to the wild type strains, whereas all mutant strains show diminished reporter activation in the region 1 stem cells. Functional studies in the embryo and the wing disc demonstrate that both wild type flies and the dp53 rescue promote cell death after irradiation, while the p53 null mutant does not. The two hp53+ strains rescued the wild type phenotype in the embryo; however, one of the hp53+ strains, named B2, was unable to induce cell death in the wing disc. The missense mutant strains do not exhibit IR-induced apoptosis in the embryo, but preliminary imaging shows they may be able to in the wing disc. We also discovered that, unlike the six hotspot mutants, wild type human p53 localizes to unidentified subnuclear compartments. Importantly, this may allow us to stratify and characterize p53 mutations according to functional differences.Item In Vivo Genome-Wide Analyses of the Drosophila p53 Transcriptional Network(2017-07-27) Kurtz, Paula S.; Fontoura, Beatriz; Abrams, John M.; Kraus, W. Lee; Krämer, Helmutp53 is the most commonly mutated gene in human cancers. Despite decades of p53 studies we do not fully understand how p53 suppresses tumors. Similar to human p53, the Drosophila counterpart is a transcription factor that can respond to genotoxic stress and promote adaptive responses at the cellular level. Our lab has leveraged the powerful genetics of Drosophila to study p53 functions in vivo. In the context of the developing fly, p53 robustly activates important apoptotic genes in response to DNA damage to promote cell death. In the embryo model, we discovered an important p53 enhancer that forms chromatin contacts through long genomic distances and enables p53 to activate various genes. How p53 programs are adapted in different cellular contexts is poorly understood. In my dissertation work I examined two layers of p53 regulation, long-range enhancer looping and p53 DNA occupancy. To further examine enhancer looping, I exploited the established embryo model and the well characterized p53 reaper enhancer. At the single cell resolution, I demonstrated that the p53 enhancer can contact multiple targets simultaneously; however these multigenic complexes appear in low frequency. I also have preliminary genome-wide data suggesting in embryos this p53 enhancer contacts additional p53 targets. In addition, through genome-scale analyses I dissected novel p53 programs in a postmitotic model (the Drosophila head). Interestingly, postmitotic p53 programs are distinct from networks described in developing cells. I found that the canonical p53 apoptotic program is unresponsive in Drosophila heads, establishing this system as an ideal in vivo model to study alternate functions of p53. To determine how p53 differential programs are specified, I tested two distinct mechanisms for tissue specific target activation, p53 enhancer looping and DNA binding. Interestingly, I observed no change in enhancer looping to cell death targets in heads. However, I did detect loss of p53 enhancer binding. Lastly, I integrated genome-wide analyses of p53 DNA occupancy and transcriptional control in embryos and heads. Interestingly, I found that at the genome-scale p53 binding landscapes poorly correlate with nearby transcriptional effects, indicating that p53 enhancers could be generally acting through long distances.Item P53 Genes Act to Restrain Mobile Elements(2015-11-24) Wylie, Annika Dawn; Amatruda, James F.; Abrams, John M.; Hobbs, Helen H.; Morrison, Sean J.Oncogenic stress provokes tumor suppression by p53 but the extent to which this regulatory axis is conserved remains unknown. Using a biosensor to visualize p53 action, we find that Drosophila p53 is selectively active in gonadal stem cells after exposure to stressors that destabilize the genome. Similar p53 activity occurred in hyperplastic growths that were triggered either by the RasV12 oncoprotein or by failed differentiation programs. In a model of transient sterility, p53 was required for the recovery of fertility after stress, and entry into the cell cycle was delayed in p53- stem cells. Together, these observations establish that the stem cell compartment of the Drosophila germline is selectively licensed for stress-induced activation of the p53 regulatory network. Furthermore, the findings uncover ancestral links between p53 and aberrant proliferation that are independent of DNA breaks and predate evolution of the ARF/Mdm2 axis. While exploring the role of p53 in this context, we made a series of observations that justify a comprehensive examination of the relationship between p53 and transposon biology. Using Drosophila, zebrafish, and mouse models, we found that p53 functions to restrict the activity of retrotransposons. Furthermore, Drosophila p53 genetically interacted with components of the piRNA pathway and, in complementation studies, normal human p53 alleles restrained these mobile elements, but mutant p53 alleles from cancer patients could not. Consistent with these results, we also found patterns of unrestrained retrotransposons in p53-driven human cancers. Together, these observations indicate that ancestral functions of p53 operate through conserved mechanisms to suppress retrotransposons. Furthermore, since human p53 mutants are disabled for this activity, our findings raise the possibility that p53 mitigates oncogenic disease, in part, by restricting retrotransposon mobility.