Mutant p53: one name, many proteins Freed-Pastor, William A; Prives, Carol
Genes & development,
06/2012, Letnik:
26, Številka:
12
Journal Article
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There is now strong evidence that mutation not only abrogates p53 tumor-suppressive functions, but in some instances can also endow mutant proteins with novel activities. Such neomorphic p53 proteins ...are capable of dramatically altering tumor cell behavior, primarily through their interactions with other cellular proteins and regulation of cancer cell transcriptional programs. Different missense mutations in p53 may confer unique activities and thereby offer insight into the mutagenic events that drive tumor progression. Here we review mechanisms by which mutant p53 exerts its cellular effects, with a particular focus on the burgeoning mutant p53 transcriptome, and discuss the biological and clinical consequences of mutant p53 gain of function.
In many organisms, the methylation of cytosine in DNA has a key role in silencing 'parasitic' DNA elements, regulating transcription and establishing cellular identity. The recent discovery that ...ten-eleven translocation (TET) proteins are 5-methylcytosine oxidases has provided several chemically plausible pathways for the reversal of DNA methylation, thus triggering a paradigm shift in our understanding of how changes in DNA methylation are coupled to cell differentiation, embryonic development and cancer.
Human embryonic stem cells (hESCs) typically exhibit “primed” pluripotency, analogous to stem cells derived from the mouse post-implantation epiblast. This has led to a search for growth conditions ...that support self-renewal of hESCs akin to hypomethylated naive epiblast cells in human pre-implantation embryos. We have discovered that reverting primed hESCs to a hypomethylated naive state or deriving a new hESC line under naive conditions results in the establishment of Stage Specific Embryonic Antigen 4 (SSEA4)-negative hESC lines with a transcriptional program resembling the human pre-implantation epiblast. In contrast, we discovered that the methylome of naive hESCs in vitro is distinct from that of the human epiblast in vivo with loss of DNA methylation at primary imprints and a lost “memory” of the methylation state of the human oocyte. This failure to recover the naive epiblast methylation landscape appears to be a consistent feature of self-renewing hypomethylated naive hESCs in vitro.
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•Reversion or derivation of hESCs in 5iLAF results in SSEA4-negative cells•SSEA4-negative hESCs show gene expression consistent with naive pluripotency•Naive hESCs show lost “memory” of gamete and blastocyst methylation•Imprinting is lost in naive hESCs
Pastor and colleagues show that reversion of primed hESCs in 5iLAF, or derivation of hESCs in 5iLAF, results in a population of naive cells characterized by loss of the marker SSEA4. However, these cells have a methylation pattern with little resemblance to blastocyst and near total loss of imprinting.
Arterial spin labeling (ASL) can be implemented by combining different labeling schemes and readout sequences. In this study, the performance of 2D and 3D single-shot pulsed-continuous ASL (pCASL) ...sequences was assessed in a group of young healthy volunteers undergoing a baseline perfusion and a functional study with a sensory-motor activation paradigm. The evaluated sequences were 2D echo-planar imaging (2D EPI), 3D single-shot fast spin-echo with in-plane spiral readout (3D FSE spiral), and 3D single-shot gradient-and-spin-echo (3D GRASE). The 3D sequences were implemented with and without the addition of an optimized background suppression (BS) scheme. Labeling efficiency, signal-to-noise ratio (SNR), and gray matter (GM) to white matter (WM) contrast ratio were assessed in baseline perfusion measurements. 3D acquisitions without BS yielded 2-fold increments in spatial SNR, but no change in temporal SNR. The addition of BS to the 3D sequences yielded a 3-fold temporal SNR increase compared to the unsuppressed sequences. 2D EPI provided better GM-to-WM contrast ratio than the 3D sequences. The analysis of functional data at the subject level showed a 3-fold increase in statistical power for the BS 3D sequences, although the improvement was attenuated at the group level. 3D without BS did not increase the maximum t-values, however, it yielded larger activation clusters than 2D. These results demonstrate that BS 3D single-shot imaging sequences improve the performance of pCASL in baseline and activation studies, particularly for individual subject analyses where the improvement in temporal SNR translates into markedly enhanced power for task activation detection.
► 3D pCASL sequences increased perfusion spatial SNR by a factor of 2. ► The addition of BS to 3D readouts increased perfusion temporal SNR by a factor of 3. ► 2D EPI showed higher GM-to-WM CBF contrast ratio. ► Statistical power to detect functional activation was higher in 3D BS sequences. ► 3D BS single-shot sequences appear as the preferable readout choice in pCASL fMRI.
Despite rapid doubling time, simple architecture and ease of metabolic labelling, a lack of genetic tools in the Lemnaceae (duckweed) has impeded the full implementation of this organism as a model ...for biological research. Here, we present technologies to facilitate high‐throughput genetic studies in duckweed. We developed a fast and efficient method for producing Lemna minor stable transgenic fronds via Agrobacterium‐mediated transformation and regeneration from tissue culture. Additionally, we engineered an artificial microRNA (amiRNA) gene silencing system. We identified a Lemna gibba endogenous miR166 precursor and used it as a backbone to produce amiRNAs. As a proof of concept we induced the silencing of CH42, a magnesium chelatase subunit, using our amiRNA platform. Expression of CH42 in transgenic L. minor fronds was significantly reduced, which resulted in reduction of chlorophyll pigmentation. The techniques presented here will enable tackling future challenges in the biology and biotechnology of Lemnaceae.
p53 is a frequent target for mutation in human tumors, and mutant p53 proteins can actively contribute to tumorigenesis. We employed a three-dimensional culture model in which nonmalignant breast ...epithelial cells form spheroids reminiscent of acinar structures found in vivo, whereas breast cancer cells display highly disorganized morphology. We found that mutant p53 depletion is sufficient to phenotypically revert breast cancer cells to a more acinar-like morphology. Genome-wide expression analysis identified the mevalonate pathway as significantly upregulated by mutant p53. Statins and sterol biosynthesis intermediates reveal that this pathway is both necessary and sufficient for the phenotypic effects of mutant p53 on breast tissue architecture. Mutant p53 associates with sterol gene promoters at least partly via SREBP transcription factors. Finally, p53 mutation correlates with highly expressed sterol biosynthesis genes in human breast tumors. These findings implicate the mevalonate pathway as a therapeutic target for tumors bearing mutations in p53.
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► Depletion of mutant p53 phenotypically reverts breast cancer cells in 3D culture ► Mutant p53 upregulates mevalonate pathway genes via SREBP transcription factors ► HMG-CoA reductase inhibitors mimic the phenotypic effects of mutant p53 depletion ► TP53 mutation correlates with elevation of mevalonate pathway genes in human tumors
Metastasis-associated p53 mutations disrupt breast tissue organization by upregulating transcription of sterol biosynthesis genes, highlighting p53 status as a predictive marker for responsiveness to statin therapy in cancer.
We recently showed that enzymes of the TET family convert 5-mC to 5-hydroxymethylcytosine (5-hmC) in DNA. 5-hmC is present at high levels in embryonic stem cells and Purkinje neurons. The methylation ...status of cytosines is typically assessed by reaction with sodium bisulfite followed by PCR amplification. Reaction with sodium bisulfite promotes cytosine deamination, whereas 5-methylcytosine (5-mC) reacts poorly with bisulfite and is resistant to deamination. Since 5-hmC reacts with bisulfite to yield cytosine 5-methylenesulfonate (CMS), we asked how DNA containing 5-hmC behaves in bisulfite sequencing.
We used synthetic oligonucleotides with different distributions of cytosine as templates for generation of DNAs containing C, 5-mC and 5-hmC. The resulting DNAs were subjected in parallel to bisulfite treatment, followed by exposure to conditions promoting cytosine deamination. The extent of conversion of 5-hmC to CMS was estimated to be 99.7%. Sequencing of PCR products showed that neither 5-mC nor 5-hmC undergo C-to-T transitions after bisulfite treatment, confirming that these two modified cytosine species are indistinguishable by the bisulfite technique. DNA in which CMS constituted a large fraction of all bases (28/201) was much less efficiently amplified than DNA in which those bases were 5-mC or uracil (the latter produced by cytosine deamination). Using a series of primer extension experiments, we traced the inefficient amplification of CMS-containing DNA to stalling of Taq polymerase at sites of CMS modification, especially when two CMS bases were either adjacent to one another or separated by 1-2 nucleotides.
We have confirmed that the widely used bisulfite sequencing technique does not distinguish between 5-mC and 5-hmC. Moreover, we show that CMS, the product of bisulfite conversion of 5-hmC, tends to stall DNA polymerases during PCR, suggesting that densely hydroxymethylated regions of DNA may be underrepresented in quantitative methylation analyses.
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