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Find video protocols related to scientific articles indexed in Pubmed.
A cisplatin-resistant head and neck cancer cell line with cytoplasmic p53(mut) exhibits ATP-binding cassette transporter upregulation and high glutathione levels.
J. Cancer Res. Clin. Oncol.
PUBLISHED: 05-16-2014
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Head and neck squamous cell carcinoma (HNSCC) cell lines with cytoplasmically sequestered mutant p53 (p53(mut_c)) are frequently more resistant to cisplatin (CDDP) than cells with mutant but nuclear p53 (p53(mut_n)). The aim of the study was to identify underlying mechanisms implicated in CDDP resistance of HNSCC cells carrying cytoplasmic p53(mut).
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Monitoring the dynamics of clonal tumour evolution in vivo using secreted luciferases.
Nat Commun
PUBLISHED: 03-04-2014
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Tumours are heterogeneous cell populations that undergo clonal evolution during tumour progression, metastasis and response to therapy. Short hairpin RNAs (shRNAs) generate stable loss-of-function phenotypes and are versatile experimental tools to explore the contribution of individual genetic alterations to clonal evolution. In these experiments tumour cells carrying shRNAs are commonly tracked with fluorescent reporters. While this works well for cell culture studies and leukaemia mouse models, fluorescent reporters are poorly suited for animals with solid tumours--the most common tumour types in cancer patients. Here we develop a toolkit that uses secreted luciferases to track the fate of two different shRNA-expressing tumour cell clones competitively, both in vitro and in vivo. We demonstrate that secreted luciferase activities can be measured robustly in the blood stream of tumour-bearing mice to accurately quantify, in a minimally invasive manner, the dynamic evolution of two genetically distinct tumour subclones in preclinical mouse models of tumour development, metastasis and therapy.
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Lung tumors on multimodal radiographs derived from grating-based X-ray imaging - A feasibility study.
Phys Med
PUBLISHED: 08-06-2013
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The purpose of this study was to assess whether grating-based X-ray imaging may have a role in imaging of pulmonary nodules on radiographs.
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Characterization of the p53 cistrome--DNA binding cooperativity dissects p53s tumor suppressor functions.
PLoS Genet.
PUBLISHED: 08-01-2013
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p53 protects us from cancer by transcriptionally regulating tumor suppressive programs designed to either prevent the development or clonal expansion of malignant cells. How p53 selects target genes in the genome in a context- and tissue-specific manner remains largely obscure. There is growing evidence that the ability of p53 to bind DNA in a cooperative manner prominently influences target gene selection with activation of the apoptosis program being completely dependent on DNA binding cooperativity. Here, we used ChIP-seq to comprehensively profile the cistrome of p53 mutants with reduced or increased cooperativity. The analysis highlighted a particular relevance of cooperativity for extending the p53 cistrome to non-canonical binding sequences characterized by deletions, spacer insertions and base mismatches. Furthermore, it revealed a striking functional separation of the cistrome on the basis of cooperativity; with low cooperativity genes being significantly enriched for cell cycle and high cooperativity genes for apoptotic functions. Importantly, expression of high but not low cooperativity genes was correlated with superior survival in breast cancer patients. Interestingly, in contrast to most p53-activated genes, p53-repressed genes did not commonly contain p53 binding elements. Nevertheless, both the degree of gene activation and repression were cooperativity-dependent, suggesting that p53-mediated gene repression is largely indirect and mediated by cooperativity-dependently transactivated gene products such as CDKN1A, E2F7 and non-coding RNAs. Since both activation of apoptosis genes with non-canonical response elements and repression of pro-survival genes are crucial for p53s apoptotic activity, the cistrome analysis comprehensively explains why p53-induced apoptosis, but not cell cycle arrest, strongly depends on the intermolecular cooperation of p53 molecules as a possible safeguard mechanism protecting from accidental cell killing.
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The pan-deacetylase inhibitor panobinostat suppresses the expression of oncogenic miRNAs in hepatocellular carcinoma cell lines.
Mol. Carcinog.
PUBLISHED: 07-31-2013
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Deacetylase inhibitors (DACi) are a new class of drugs with a broad spectrum of mechanisms that favor their application in cancer therapy. Currently, the exact mechanisms and cellular effects of DACi have not been fully elucidated. In addition to their effects on histone acetylation, DACi can interfere with gene expression via miRNA pathways. Treatment with panobinostat (LBH589), a novel potent DACi, led to the highly aberrant modulation of several miRNAs in hepatocellular carcinoma (HCC) cell lines as shown by miRNA array analysis. Among them, hsa-miR-19a, hsa-miR-19b1 and the corresponding precursors were down-regulated by panobinostat in TP53(-/-) Hep3B and TP53(+/+) HepG2 cell lines; hsa-miR30a-5p mature form only was suppressed in both HCC cell lines, as confirmed by further RT-qPCR analysis. In HCC cell lines, panobinostat caused the upregulation of the predicted miRNA targets APAF1 and Beclin1 protein levels. Transfection with oligonucleotides mimicking these miRNAs led to an increase in the viability rate of both cell lines as analyzed by impedance-based real-time cell analysis. In addition, transfecting miRNA mimicking oligonucleotides resulted in the decrease of APAF1, Beclin1 and PAK6 at the protein level, proving the regulating influence of the investigated miRNAs on gene final products. The overexpression of the above mentioned oncomiRs in Hep3B and HepG2 cell lines leads to cell proliferation and downregulation of cell death associated proteins. In our model, panobinostat exerts its anti-cancer effect by suppressing these miRNAs and restoring the expression of their corresponding tumor suppressor targets. © 2013 Wiley Periodicals, Inc.
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A senescence-inflammatory switch from cancer-inhibitory to cancer-promoting mechanism.
Cancer Cell
PUBLISHED: 04-19-2013
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Senescence, perceived as a cancer barrier, is paradoxically associated with inflammation, which promotes tumorigenesis. Here, we characterize a distinct low-grade inflammatory process in stressed epithelium that is related to para-inflammation; this process either represses or promotes tumorigenesis, depending on p53 activity. Csnk1a1 (CKI?) downregulation induces a senescence-associated inflammatory response (SIR) with growth arrest in colorectal tumors, which loses its growth control capacity in the absence of p53 and instead, accelerates growth and invasiveness. Corresponding processes occur in CKI?-deleted intestinal organoids, assuming tumorigenic transformation properties ex vivo, upon p53 loss. Treatment of organoids and mice with anti-inflammatory agents suppresses the SIR and prevents p53-deficient organoid transformation and mouse carcinogenesis. SIR/para-inflammation suppression may therefore constitute a key mechanism in the anticarcinogenic effects of nonsteroidal anti-inflammatory drugs.
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p53 DNA binding cooperativity is essential for apoptosis and tumor suppression in vivo.
Cell Rep
PUBLISHED: 03-12-2013
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Four molecules of the tumor suppressor p53 assemble to cooperatively bind proapoptotic target genes. The structural basis for cooperativity consists of interactions between adjacent DNA binding domains. Mutations at the interaction interface that compromise cooperativity were identified in cancer patients, suggesting a requirement of cooperativity for tumor suppression. We report on an analysis of cooperativity mutant p53E177R mice. Apoptotic functions of p53 triggered by DNA damage and oncogenes were abolished in these mice, whereas functions in cell-cycle control, senescence, metabolism, and antioxidant defense were retained and were sufficient to suppress development of spontaneous T cell lymphoma. Cooperativity mutant mice are nevertheless highly cancer prone and susceptible to different oncogene-induced tumors. Our data underscore the relevance of DNA binding cooperativity for p53-dependent apoptosis and tumor suppression and highlight cooperativity mutations as a class of p53 mutations that result in a selective loss of apoptotic functions due to an altered quaternary structure of the p53 tetramer.
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MicroRNA29a regulates the expression of the nuclear oncogene Ski.
Blood
PUBLISHED: 06-17-2011
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MicroRNAs (miRNAs) are small, noncoding RNA molecules that regulate growth and differentiation. miRNAs are frequently located at cancer-specific fragile sites in the human genome, such as chromosome 7q. The nuclear oncogene SKI is up-regulated in acute myeloid leukemia (AML) with -7/del7q. Here we asked whether loss of miRNAs on chromosome 7q may explain this up-regulation. miR-29a expression was found to be down-regulated in AML with -7/del7q. Forced expression of miR-29a down-regulated Ski and its target gene, Nr-CAM, whereas miR-29a inhibition induced Ski expression. Luciferase assays validated a functional binding site for miR-29a in the 3 untranslated region of SKI. Finally, in samples of AML patients, we observed an inverse correlation of Ski and miR-29a expression, respectively. In conclusion, up-regulation of Ski in AML with -7/del7q is caused by loss of miR-29a. miR-29a may therefore function as an important tumor suppressor in AML by restraining expression of the SKI oncogene.
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Targeting p73 in cancer.
Cancer Lett.
PUBLISHED: 06-03-2011
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p73 is a member of the p53 family of tumor suppressors. Transactivating isoforms of p73 (TAp73) have p53-like, anti-proliferative and pro-apoptotic activities that are crucial for an efficient chemotherapy response. In line with this, genetic studies in mice have confirmed that TAp73 acts as a tumor suppressor. However, in contrast to p53, which is commonly inactivated in human cancer by point mutations, the TP73 gene is almost never mutated. Instead, the tumor suppressor activity of TAp73 is inhibited through a variety of mechanisms including epigenetic silencing and complex formation with inhibitory proteins. All these mechanisms have in common that they are in principle reversible and therefore amenable to therapeutic intervention. Here, we will review how tumor cells control the tumor suppressor activity of TAp73 and discuss possible strategies targeting p73 for reactivation.
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Life or death: p53-induced apoptosis requires DNA binding cooperativity.
Cell Cycle
PUBLISHED: 10-11-2010
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The tumor suppressor p53 provides exquisite protection from cancer by balancing cell survival and death in response to stress. Sustained stress or irreparable damage trigger p53s killer functions to permanently eliminate genetically-altered cells as a potential source of cancer. To prevent the unnecessary loss of cells that could cause premature aging as a result of stem cell attrition, the killer functions of p53 are tightly regulated and balanced against protector functions that promote damage repair and support survival in response to low stress or mild damage. In molecular terms these p53-based cell fate decisions involve protein interactions with cofactors and modifying enzymes, which modulate the activation of distinct sets of p53 target genes. In addition, we demonstrate that part of this regulation occurs at the level of DNA binding. We show that the killer function of p53 requires the four DNA binding domains within the p53 tetramer to interact with one another. These intermolecular interactions enable cooperative binding of p53 to less perfect response elements in the genome, which are present in many target genes essential for apoptosis. Modulating p53 interactions within the tetramer could therefore present a novel promising strategy to fine-tune p53-based cell fate decisions.
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Malignant transformation in a defined genetic background: proteome changes displayed by 2D-PAGE.
Mol. Cancer
PUBLISHED: 03-26-2010
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Cancer arises from normal cells through the stepwise accumulation of genetic alterations. Cancer development can be studied by direct genetic manipulation within experimental models of tumorigenesis. Thereby, confusion by the genetic heterogeneity of patients can be circumvented. Moreover, identification of the critical changes that convert a pre-malignant cell into a metastatic, therapy resistant tumor cell, however, is one necessary step to develop effective and selective anti-cancer drugs. Thus, for the current study a cell culture model for malignant transformation was used: Primary human fibroblasts of the BJ strain were sequentially transduced with retroviral vectors encoding the genes for hTERT (cell line BJ-T), simian virus 40 early region (SV40 ER, cell line BJ-TE) and H-Ras V12 (cell line BJ-TER).
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DNA binding cooperativity of p53 modulates the decision between cell-cycle arrest and apoptosis.
Mol. Cell
PUBLISHED: 02-16-2010
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p53 limits the proliferation of precancerous cells by inducing cell-cycle arrest or apoptosis. How the decision between survival and death is made at the level of p53 binding to target promoters remains unclear. Using cancer cell lines, we show that the cooperative nature of DNA binding extends the binding spectrum of p53 to degenerate response elements in proapoptotic genes. Mutational inactivation of cooperativity therefore does not compromise the cell-cycle arrest response but strongly reduces binding of p53 to multiple proapoptotic gene promoters (BAX, PUMA, NOXA, CASP1). Vice versa, engineered mutants with increased cooperativity show enhanced binding to proapoptotic genes, which shifts the cellular response to cell death. Furthermore, the cooperativity of DNA binding determines the extent of apoptosis in response to DNA damage. Because mutations, which impair cooperativity, are genetically linked to cancer susceptibility in patients, DNA binding cooperativity contributes to p53s tumor suppressor activity.
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Oncogenic RAS enables DNA damage- and p53-dependent differentiation of acute myeloid leukemia cells in response to chemotherapy.
PLoS ONE
PUBLISHED: 04-26-2009
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Acute myeloid leukemia (AML) is a clonal disease originating from myeloid progenitor cells with a heterogeneous genetic background. High-dose cytarabine is used as the standard consolidation chemotherapy. Oncogenic RAS mutations are frequently observed in AML, and are associated with beneficial response to cytarabine. Why AML-patients with oncogenic RAS benefit most from high-dose cytarabine post-remission therapy is not well understood. Here we used bone marrow cells expressing a conditional MLL-ENL-ER oncogene to investigate the interaction of oncogenic RAS and chemotherapeutic agents. We show that oncogenic RAS synergizes with cytotoxic agents such as cytarabine in activation of DNA damage checkpoints, resulting in a p53-dependent genetic program that reduces clonogenicity and increases myeloid differentiation. Our data can explain the beneficial effects observed for AML patients with oncogenic RAS treated with higher dosages of cytarabine and suggest that induction of p53-dependent differentiation, e.g. by interfering with Mdm2-mediated degradation, may be a rational approach to increase cure rate in response to chemotherapy. The data also support the notion that the therapeutic success of cytotoxic drugs may depend on their ability to promote the differentiation of tumor-initiating cells.
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Induction of early growth response-1 mediates microglia activation in vitro but is dispensable in vivo.
Neuromolecular Med.
PUBLISHED: 02-11-2009
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We have previously identified activation of microglia and induction of the early growth response gene 1 (Egr1) in the retina of retinoschisin-deficient (Rs1h(-/Y)) mice. We hypothesized that microglial expression of Egr1 might support retinal microgliosis. To test this, Egr1 transcript levels were determined in RNAs isolated from early postnatal retinas and primary microglia from Rs1h(-/Y) mice and wild-type controls. Egr1 mRNA expression was strongly induced in retinoschisin-deficient retinas as well as in ex vivo isolated microglia. Increased microglial Egr1 protein expression was concordantly detected in retinal sections of Rs1h(-/Y) mice using immunohistochemistry. Prominent activation-dependent Egr1 mRNA and protein expression was also confirmed in murine BV-2 microglia. Using binding site prediction and chromatin immunoprecipitation, we identified that the Egr1 promoter itself and the microglial marker genes Clec7a and Caspase11 are direct transcriptional targets of Egr1. Over-expression of Egr1 in BV-2 cells by adenoviral infection promoted Clec7a and Caspase11 mRNA synthesis, whereas expression of the Egr1 repressor NAB2 blocked the transcription of these genes. To analyze whether Egr1 was absolutely required for microglial marker expression in vivo, transcript levels were quantified in Rs1h(-/Y)/Egr1(-/-) retinas. No significant differences in activation marker expression could be measured in retinal tissue from Rs1h(-/Y)/Egr1(-/-) mice compared to Rs1h(-/Y) mice, suggesting that lack of Egr1 does not impair transcription of microglia genes in vivo. Taken together, our findings suggest that increased Egr1 expression is present in activated retinal microglia and contributes to their activation. However, up-regulation of Egr1 is not absolutely required for retinal microglia activation in vivo.
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p53 and p73 in suppression of Myc-driven lymphomagenesis.
Int. J. Cancer
PUBLISHED: 01-16-2009
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Induction of apoptosis by the tumor suppressor p53 is known to protect from Myc-driven lymphomagenesis. The p53 family member p73 is also a proapoptotic protein, which is activated in response to oncogenes like Myc. Here, we have investigated whether p73 provides a similar protection from Myc-driven lymphomas as p53. Confirming previous studies, the inactivation of a single p53 allele (p53+/-) strongly reduced the median survival of Emu-Myc transgenic mice from 103 to 39 days and was invariably associated with a loss of the wild-type p53 allele. In contrast, mutational inactivation of a p73 allele (p73+/-) reduced the median survival by only 12 days. Lymphomas that developed in the p73+/- background showed no loss of heterozygosity (LOH). Furthermore, gene expression profiling of p73+/+, p73+/- and p73-/- lymphomas indicated that p73+/- lymphomas retained p73 transcriptional activity. Subtle gene expression differences between p73+/+ and p73+/- lymphomas, however, suggest a haploinsufficient phenotype on some p73 target genes. This might help to explain why p73+/- animals succumbed to disease slightly earlier than their p73+/+ littermates (log-rank test p<0.0395) and why p73 often shows monoallelic inactivation in human lymphomas. Together these data demonstrate that in Myc-driven lymphomagenesis p73 has weak tumor suppressor activity compared with p53.
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iTRAQ analysis of a cell culture model for malignant transformation, including comparison with 2D-PAGE and SILAC.
J. Proteome Res.
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To study human cancer development, cell culture models for malignant transformation can be used. In 1999 Hahn and Coworkers introduced such a model system and established herewith a basis for research on human tumorigenesis. Primary human fibroblasts are sequentially transduced with defined genetic elements (hTERT, SV40 ER, and H-RasV12), resulting in four defined cell lines, whereby the last has a fully transformed phenotype. In order to get a deeper insight into the molecular biology of human tumorigenesis, we compared the proteomes of these four cell lines following a multimethod concept. At the beginning we assumed SILAC and sample fractionation with COFRADIC is the method of choice to analyze the cell culture model for malignant transformation. Here, the compared samples are combined before sample preparation, thus avoiding differences in sample preparation, and using COFRADIC notably reduces sample complexity. Because 2D-PAGE is a standard method for the separation and visualization of closely related proteomes, we decided to analyze and compare the proteomes of these four cell lines in a first approach by differential 2D-PAGE. Surprisingly, we discovered much more unique results with iTRAQ and sample fractionation with SCX than with the combination of 2D-PAGE and SILAC-COFRADIC. Moreover, iTRAQ outperforms the other strategies not only in number of yielded results but also in analysis time. Here, we present the iTRAQ quantification results and compare them with the results of 2D-PAGE and SILAC-COFRADIC. We found changes in the protein level at each transition. Thereby, SV40 has the strongest impact on the proteome. In detail we identified 201 regulated proteins. Beside others, these proteins are involved in cytoskeleton, RNA processing, and cell cycle, such as CDC2, hnRNPs, snRNPs, collagens, and MCM proteins. For example, MCM proteins are up-regulated and collagens are down-regulated due to SV40 ER expression. Furthermore we made the observation that proteins containing the same domain have analogous regulation profiles during malignant transformation. For instance, several proteins containing a CH or LIM domain are down-regulated. Moreover, by this study and the defined cell culture model, changes could be clearly matched to specific steps during tumorigenesis.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.