Questo lavoro è stato sostenuto da una sovvenzione da New Innovator Award Program del direttore NIH (ED). AA è stato sostenuto in parte da premi di laurea dal National Cancer Institute e la National Science Foundation.

1. Rendere i vettori virali Le sequenze codificanti per il gene di interesse, così come i controlli positivi e negativi, sono inseriti in pBABEpuro con metodi tradizionali di clonazione (amplificazione PCR, digestione di restrizione e legatura). Ci sono quattro siti di restrizione sul vettore in cui il DNA può essere inserito: BamHI, SnaBI, EcoRI e Sali. <ol><li> Preparare trasfezione-grade plasmide DNA dalle cellule competenti mediante il kit plasmide midi QIAGEN. </li><li> Misurare la concentrazione di DNA utilizzando uno spettrofotometro NanoDrop2000. </li></ol> 2. Retrovirus Produzione La linea cellulare di packaging Plat-E sarà utilizzato per produrre retrovirus ecotropic che porterà il cDNA di interesse per cellule NIH 3T3. <ol><li> Stabilire Plat-E culture di cellule congelate <ol><li> Scongelare rapidamente cellule Plat-E in un bagno d&#39;acqua ° C 37 e trasferire in una provetta da 15 ml. Lentamente aggiungere 9 ml di Plat-E medio (Dudi lbecco modificato (DMEM) + 10% siero fetale bovino (FBS) + penicillina e streptomicina). </li><li> Centrifugare la provetta a 180 x g per 5 minuti e scartare il surnatante. </li><li> Risospendere le cellule con 10 ml di Plat-E a medio e trasferimento in un piatto di cultura 10 cm. </li><li> Incubare le cellule a 37 ° C, 5% CO 2 incubatore fino a quando sono 80-90% confluenti (circa 2 giorni). </li></ol></li><li> Splitting cellulare <ol><li> Aspirare il medio e lavare le cellule una volta con PBS. </li><li> Aggiungere 2 ml di 0,05% tripsina-EDTA e incubare per 1 min a RT. </li><li> Staccare le cellule finger tapping, aggiungere 10 ml di Plat-E di media, e trasferire la sospensione cellulare in una provetta da 15 ml. </li><li> Centrifugare la provetta a 180 x g per 5 minuti e scartare il surnatante. </li><li> Risospendere le cellule con 10 ml di Plat-E a medio e sementi in nuovi piatti a 1: 4-1: 6 diluizioni. </li></ol></li><li> Plat-E Seeding e Transfection <ol><li> Seed 2 x 10 6cellule per 10 centimetri piatto di coltura usando Plat-E medio senza antibiotici. </li><li> Incubare le cellule O / N a 37 ° C, 5% CO 2 incubatore. </li><li> Il giorno dopo, il trasferimento di 300 ml di Opti-MEM in 1,5 ml provetta da microcentrifuga. </li><li> Aggiungere 27 ml di polyethylenimine (PEI), un costo-efficacia di trasfezione reattivo 12, al tubo preparato con Opti-MEM. Mescolare delicatamente con le dita toccando e incubare per 5 minuti a temperatura ambiente. </li><li> Aggiungere 9 mg di trasfezione-grade plasmide DNA nel tubo Opti-MEM / PEI, mescolare delicatamente con il vortex e incubare per 15 minuti a temperatura ambiente. </li><li> Aggiungere il DNA / PEI goccia a goccia complesso nel piatto Plat-E, e incubare O / N a 37 ° C, 5% CO 2. </li><li> Il giorno dopo, aspirare il supporto contenente il reagente di trasfezione e aggiungere 10 ml di fresco medio Plat-E (senza antibiotici). </li><li> Riportare le cellule per l&#39;incubatore. </li></ol></li></ol> 3. Cellule NIH 3T3 e infezioni <ol><li> Stabilire NIHCulture 3T3 e il seeding <ol><li> Scongelare rapidamente cellule NIH 3T3 in ° C bagnomaria 37 e trasferire in una provetta da 15 ml. Aggiungere lentamente 9 ml di NIH 3T3 medium (DMEM + 10% FBS). </li><li> Centrifugare la provetta a 180 x g per 5 minuti e scartare il surnatante. </li><li> Risospendere le cellule con 10 ml di NIH 3T3 media e il trasferimento ad un piatto di cultura 10 cm. </li><li> Incubare le cellule a 37 ° C, 5% CO 2 incubatore. È molto importante che le cellule NIH 3T3 sono sempre tenute underconfluent (50-60%), la frequenza di trasformazione spontanea (e quindi i livelli basali di formazione foci) aumenta una volta coltura raggiunga confluenza. </li><li> Seed 3 x 10 5 cellule per 10 centimetri piatto e incubare O / N per l&#39;infezione il giorno successivo. </li></ol></li><li> Infezione <ol><li> Raccogliere il supernatante retrovirale dal piatto Plat-E utilizzando una siringa monouso 10 ml, filtrare attraverso un filtro a membrana di nylon dimensioni 0,45 micron pori, e trasferirlo in un 15 mltube. </li><li> Aggiungere 10 ml di fresco medio Plat-E per le cellule (senza antibiotici), e restituirli alla incubatrice. </li><li> Aspirare il supporto dal piatto delle cellule NIH 3T3 di essere infetti, e aggiungere 5 ml di regolare medie NIH 3T3 e 5 ml di virus contenenti surnatante filtrato. </li><li> Aggiungere polibrene al piatto ad una concentrazione di 6 mg / ml. </li><li> Incubare le cellule O / N a 37 ° C, 5% CO 2. </li><li> Il giorno dopo, ripetere i punti 3.2.1 - 3.2.5 per un secondo turno di infezione. </li><li> Sostituire il supporto contenente il virus, con regolare medio NIH 3T3. </li><li> Lasciare le cellule NIH 3T3 a crescere per 2-3 settimane, sostituendo la soluzione, se necessario. Controllare espressione della proteina di interesse mediante elettroforesi delle proteine ​​convenzionale e immunoblot su lisati cellulari interi da lastre di replica. </li></ol></li></ol> 4. Colorazione e quantificazione <ol><li> Cristallo viola colorazione <ol><li> Aspirare il mezzo di NIH 3T3 e posizionare i piattisul ghiaccio. </li><li> Lavare i piatti due volte con PBS freddo. </li><li> Fissare le cellule con metanolo ghiacciata per 10 min. </li><li> Togliere i piatti da ghiaccio, aspirare il metanolo, e aggiungere 3 ml di soluzione di cristallo viola 0,5% effettuati nel 25% di metanolo (RT). </li><li> Incubare i piatti per 5 minuti a RT. </li><li> Aspirare la soluzione a cristalli viola e risciacquare accuratamente il piatto con Milli-Q H 2 O fino a quando nessun colore si stacca nel risciacquo. </li><li> Lasciare i piatti asciugare O / N su un banco (scoperto). </li></ol></li><li> Foci Quantificazione <ol><li> Una volta che i piatti sono asciutti, utilizzare un righello per misurare le collezioni di colore scuro di cellule sulla piastra. Contano solo quelli che sono più di 5 mm di diametro come &quot;focolai&quot;. Registrare il numero di focolai e calcolare le medie e il significato rispetto ai controlli. </li></ol></li></ol>

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La FFA fornisce un metodo semplice e veloce per valutare trasformazione maligna in vitro. E &#39;suscettibile di proiezione di un numero relativamente elevato di geni candidati, e le sue esigenze tecniche modeste rendono conveniente. Inoltre, due o più geni possono essere coexpressed (talvolta indicato come un saggio &quot;cooperazione&quot;) per valutare il potenziale cancerogeno della combinazione. I vantaggi di questo test si basano sulla sua tecnica semplice, la facilità di quantificazione e la sua relati...

Le cellule tumorali possono essere distinti dalle loro controparti normali da una vasta gamma di alterazioni, dai modelli di espressione genica per epigenomica ai cambiamenti morfologici e proliferativi. Tra questi ultimi, ridotta dipendenza da siero, perdita di contatto (densità) inibizione, l&#39;acquisizione della proliferazione di ancoraggio-indipendente e, in ultima analisi, la capacità di formare tumori quando iniettate in animali sono utili, indicatori misurabili di trasformazione maligna 3. Diversi in vitro e in vivo sono stati sviluppati per la trasformazione cellulare. In vitro mirare a individuare e cambiamenti nella cultura morfologia (attenzione test formazione), la dinamica della cultura (il tasso di crescita, la densità di saturazione) e fattore di crescita di misura (la crescita nel siero ridotta) o ancoraggio (crescita in soft agar) esigenze. Il gold standard per determinare la natura maligna di un tipo di cellula rimane la formazione di tumori (xenotrapianti) negli animali da esperimento. Tuttavia, tl costo e la durata degli studi in vivo non sempre li rendono giustificabile in una prima fase di validazione o proiezione di oncogeni candidati. Anche se nessun test in vitro fornisce una valutazione definitiva del potenziale oncogeno di un gene, essi forniscono informazioni in potenziale oncogeno che potrebbero restringere futuro studi in vivo. Uno dei sistemi più utilizzati per valutare il potenziale oncogeno in vitro è al centro di Formazione Assay 2. Questo approccio si basa sull&#39;uso di NIH 3T3 fibroblasti di topo, una linea cellulare non trasformato che mostra forte inibizione contatto. Sovraespressione di un oncogene si traduce in perdita di crescita di densità-dipendente; cellule trasformate possono crescere in strati multipli, formando &quot;fuochi&quot;, facilmente visualizzato sullo sfondo monostrato di cellule non trasformate. La formazione fuoco Assay, poi, misura la capacità di un oncogene candidato per indurre la trasformazione maligna, come dimostra la perdita di contatto inibition come un fenotipo misurabile. L&#39;FFA è stato utilizzato per valutare la trasformazione da sovraespressione di proteine ​​chinasi (ad esempio, Src 4, BRAF 5), fattori di trascrizione (ad esempio, N-myc 6) recettori, G-accoppiati alle proteine ​​(ad esempio, P2RY8 7) e GTPases (ad esempio, , Ras 1), tra gli altri. La relativa facilità di questo saggio rende una buona scelta che fornirà una risposta rapida e visivamente chiaro se sovraespressione del gene è sufficiente per trasformare cellule di fibroblasti di topo NIH 3T3 in vitro. L&#39;FFA descritto in questo protocollo utilizza la linea cellulare di confezionamento Plat-E 8, che fornisce proteine ​​virali di imballaggio, e il vettore retrovirale pBABEpuro 9 (Addgene plasmide 1764) per la produzione di retrovirus. Dopo trasfezione con il costrutto pBABEpuro contenente il gene di interesse, la linea cellulare Plat-E produrrà retrovirus ecotropic che può essere utilizzato per infettare cellule NIH 3T3. Til suo metodo virale di consegna del gene è più efficiente rispetto ai metodi di trasfezione chimici tradizionali e offre un modo per esprimere in modo sostenibile il gene 10. Una volta inseriti nel genoma delle cellule NIH 3T3, sovraespressione del gene di interesse è guidato dalla lunghe ripetizioni terminali virali (LTR) promotore 11. Questa costante espressione può essere usato per determinare se il gene di interesse ha attività oncogenica, come misurato dalla formazione di foci, sulle cellule NIH 3T3.

<table><tbody><tr><td>pBABE-puro vector</td><td>Addgene</td><td>Plasmid 1764</td><td>cloning vector</td></tr><tr><td>Platinum-E Retroviral Packaging Cell Line, Ecotropic</td><td>Cell Biolabs, Inc.</td><td>RV-101</td><td>cell line for viral production</td></tr><tr><td>NIH 3T3 Cell Line murine</td><td>Sigma-Aldrich</td><td>93061524</td><td>cell line for focus formation assay</td></tr><tr><td>10 ml BD Luer-Lok tip syringe</td><td>BD Biosciences</td><td>309604</td><td>viral production reagent</td></tr><tr><td>0.45 &amp;mu;m Puradisc Syringe Filter</td><td>Whatman</td><td>6750-2504</td><td>viral production reagent</td></tr><tr><td>Polyethylenimine (PEI)</td><td>Polysciences, Inc.</td><td>23966-2</td><td>cell transfection reagent</td></tr><tr><td>Polybrene Infection / Transfection Reagent</td><td>EMD Millipore</td><td>TR-1003-G</td><td>cell transfection reagent</td></tr><tr><td>Crystal Violet</td><td>Fisher Scientific</td><td>C581-25</td><td>cell stain reagent</td></tr><tr><td>Plasmid Plus Midi Kit</td><td>QIAGEN</td><td>12945</td><td>plasmid purification</td></tr><tr><td>BD Falcon Tissue Culture Dishes</td><td>BD Biosciences</td><td>353003</td><td>cell culture supplies</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Gibco</td><td>11995-065</td><td>cell culture media</td></tr><tr><td>0.05% Trypsin-EDTA</td><td>Gibco</td><td>25300-054</td><td>cell culture supplies</td></tr><tr><td>Opti-MEM I Reduced Serum Medium</td><td>Gibco</td><td>31985-062</td><td>cell culture media</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco</td><td>16000-044</td><td>cell culture media</td></tr></tbody></table>

focus formation: a cell-based assay to determine the oncogenic potential of a gene

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent growth potential, and the ability to form tumors in experimental animals1. In NIH 3T3 cells, the Ras signal transduction pathway is known to trigger many of these events, what is known as Ras transformation. The introduction of an overexpressed gene in NIH 3T3 cells may promote morphological transformation and loss of contact inhibition, which can help determine the oncogenic potential of that gene of interest. An assay that provides a straightforward method to assess one aspect of the transforming potential of an oncogene is the Focus Formation Assay (FFA)2. When NIH 3T3 cells divide normally in culture, they do so until they reach a confluent monolayer. However, in the presence of an overexpressed oncogene, these cells can begin to grow in dense, multilayered foci1 that can be visualized and quantified by crystal violet or Hema 3 staining. In this article we describe the FFA protocol with retroviral transduction of the gene of interest into NIH 3T3 cells, and how to quantify the number of foci through staining. Retroviral transduction offers a more efficient method of gene delivery than transfection, and the use of an ecotropic murine retrovirus provides a biosafety control when working with potential human oncogenes.

MXD3 è un fattore fondamentale cerniera elica-ansa-elica leucina (bHLHZ) di trascrizione che è un membro del MYC / MAX / network MAD. Si tratta di un atipico membro della famiglia MAD 13-15, ed è stato segnalato per essere coinvolti nella carcinogenesi 16,17. Rispetto al pBABEpuro (controllo negativo) e MYC (controllo positivo), i piatti NIH 3T3 dove era sovraespresso MXD3 era significativamente inferiore foci (Figura 1A). I dati della figura 1B sono stati racco...

Watch this Scientific Journal Video about Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene at JoVE.com

Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

Il saggio Formazione Focus fornisce un metodo semplice per valutare il potenziale di trasformare un oncogene candidato.

Concentrarsi Formazione: Un test cellulare per determinare il potenziale oncogeno di un gene

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent ...

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27.8K Views.  University of California, Davis.  Il saggio Formazione Focus fornisce un metodo semplice per valutare il potenziale di trasformare un oncogene candidato. 

Video: Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery of the next generation of novel drug targets. It is becoming apparent that more complex models of cancer are required to fully appreciate the contributing factors that drive tumorigenesis in vivo and increase the efficacy of novel therapies that make the transition from pre-clinical models to clinical trials.
Here we present a methodology for generating uniform and reproducible tumor spheroids that can be subjected to siRNA functional screening. These spheroids display many characteristics that are found in solid tumors that are not present in traditional two-dimension culture. We show that several commonly used breast cancer cell lines are amenable to this protocol. Furthermore, we provide proof-of-principle data utilizing the breast cancer cell line BT474, confirming their dependency on amplification of the epidermal growth factor receptor HER2 and mutation of phosphatidylinositol-4,5-biphosphate 3-kinase (PIK3CA) when grown as tumor spheroids. Finally, we are able to further investigate and confirm the spatial impact of these dependencies using immunohistochemistry.

Identifying novel drug targets that transition from pre-clinical testing to human trials is a scientific priority. To that end, here we describe a functional genomics approach for examining the impact of gene depletion on cancer cell line spheroids, which more appropriately model human cancers in vivo.

Utilizzando Genomica Funzionale screening per identificare gli obiettivi farmaco potenzialmente romanzo in Cancer Cell Culture Spheroid

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

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Colony Forming Cell (CFC) Assay for Human Hematopoietic Cells

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and leukemogenesis. They have the capacity to differentiate into lymphoid and myeloid lineages. The colony forming cell (CFC) assay is used to study the proliferation and differentiation pattern of hematopoietic progenitors by their ability to form colonies in a semisolid medium. The number and the morphology of the colonies formed by a fixed number of input cells provide preliminary information about the ability of progenitors to differentiate and proliferate. Cells can be harvested from individual colonies or from the whole plate to further assess their numbers and differentiation states using flow cytometry and morphologic evaluation of Giemsa-stained slides. This assay is useful for assessing myeloid but not lymphoid differentiation. The term myeloid in this context is used in its wider sense to encompass granulocytic, monocytic, erythroid, and megakaryocytic lineages.

We have used this assay to assess the effects of oncogenes on the differentiation of primary human CD34+ cells derived from peripheral blood. For this purpose cells are transduced with either control retroviral construct or a construct expressing the oncogene of interest, in this case NUP98-HOXA9. We employ a commonly used retroviral vector, MSCV-IRES-GFP, that expresses a bicistronic mRNA that produces the gene of interest and a GFP marker. Cells are pre-activated by growing in the presence of cytokines for two days prior to retroviral transduction. After another two days, GFP+ cells are isolated by fluorescence-activated cell sorting (FACS) and mixed with a methylcellulose-containing semisolid medium supplemented with cytokines and incubated till colonies appear on the surface, typically 14 days. The number and morphology of the colonies are documented. Cells are then removed from the plates, washed, counted, and subjected to flow cytometry and morphologic examination. Flow cytometry with antibodies specific to the cell surface markers expressed during hematopoiesis provides information about lineage and maturation stage. Morphological studies of individual cells under a microscope after Wright- Giemsa staining provide further information with regard to lineage and maturation. Comparison of cells transduced with control empty vector to those transduced with an oncogene reveals the effects of the oncogene on hematopoietic differentiation.

Colony Forming Cell CFC Assay for Human Hematopoietic Cells

The colony forming cell (CFC) assay is an in vitro assay in which hematopoietic progenitors form colonies in a semi-solid medium. A combination of colony morphology, cell morphology, and flow cytometry are used to assess the ability of the progenitors to proliferate and differentiate along the different hematopoietic lineages.

Formanti colonia cellulare (CFC) Analisi per cellule emopoietiche

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and ...

Immunologia e infezioni

Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, a small population of cells does not respond to TKI treatment, resulting in minimal residual disease (MRD); even the most potent TKIs fail to eradicate these cells. These MRD cells serve as a reservoir to develop resistance to therapy. Why TKI treatment is ineffective against MRD cells is not known. Growth factor signaling is implicated in supporting the survival of MRD cells during TKI treatment, but a mechanistic understanding is lacking. Recent studies demonstrated that an elevated c-Fos and Dusp1 expression as a result of convergent oncogenic and growth factor signaling in MRD cells mediate TKI resistance. The genetic and chemical inhibition of c-Fos and Dusp1 renders CML exquisitely sensitive to TKIs and cures CML in both genetic and humanized mouse models. We identified these target genes using multiple microarrays from TKI-sensitive and -resistant cells. Here, we provide methods for target validation using in vitro and in vivo mouse models. These methods can easily be applied to any target for genetic validation and therapeutic development.

Here, we describe protocols for the genetic and chemical validation of c-Fos and Dusp1 as a drug target in leukemia using in vitro and in vivo genetic and humanized mouse models. This method can be applied to any target for genetic validation and therapeutic development.

Metodi per valutare il ruolo di c-Fos e Dusp1 nella dipendenza dell'Oncogene

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, ...

Yeast As a Chassis for Developing Functional Assays to Study Human P53

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of different functional assays to study the impacts of 1) binding site [i.e., response element (RE)] sequence variants on P53 transactivation specificity or 2) TP53 mutations, co-expressed cofactors, or small molecules on P53 transactivation activity. Different basic and translational research applications have been developed. Experimentally, these approaches exploit two major advantages of the yeast model. On one hand, the ease of genome editing enables quick construction of qualitative or quantitative reporter systems by exploiting isogenic strains that differ only at the level of a specific P53-RE to investigate sequence-specificity of P53-dependent transactivation. On the other hand, the availability of regulated systems for ectopic P53 expression allows the evaluation of transactivation in a wide range of protein expression. Reviewed in this report are extensively used systems that are based on color reporter genes, luciferase, and the growth of yeast to illustrate their main methodological steps and to critically assess their predictive power. Moreover, the extreme versatility of these approaches can be easily exploited to study different TFs including P63 and P73, which are other members of TP53 gene family.

Presented here are four protocols to construct and exploit yeast Saccharomyces cerevisiae reporter strains to study human P53 transactivation potential, impacts of its various cancer-associated mutations, co-expressed interacting proteins, and the effects of specific small molecules.

Il lievito come telaio per lo sviluppo di saggi funzionali per studiare l'Uomo P53

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of ...

Oncogenic Gene Fusion Detection Using Anchored Multiplex Polymerase Chain Reaction Followed by Next Generation Sequencing

Gene fusions frequently contribute to the oncogenic phenotype of many different types of cancer. Additionally, the presence of certain fusions in samples from cancer patients often directly influences diagnosis, prognosis, and/or therapy selection. As a result, the accurate detection of gene fusions has become a critical component of clinical management for many disease types. Until recently, clinical gene fusion detection was predominantly accomplished through the use of single-gene assays. However, the ever-growing list of gene fusions with clinical significance has created a need for assessing fusion status of multiple genes simultaneously. Next generation sequencing (NGS)-based testing has met this demand through the ability to sequence nucleic acid in massively parallel fashion. Multiple NGS-based approaches that employ different strategies for gene target enrichment are now available for use in clinical molecular diagnostics, each with its own strengths and weaknesses. This article describes the use of anchored multiplex PCR (AMP)-based target enrichment and library preparation followed by NGS to assess for gene fusions in clinical solid tumor specimens. AMP is unique among amplicon-based enrichment approaches in that it identifies gene fusions regardless of the identity of the fusion partner. Detailed here are both the wet-bench and data analysis steps that ensure accurate gene fusion detection from clinical samples.

This article details the use of an anchored multiplex polymerase chain reaction-based library preparation kit followed by next-generation sequencing to assess for oncogenic gene fusions in clinical solid tumor samples. Both wet-bench and data analysis steps are described.

Rilevamento oncogenico della fusione genica utilizzando la reazione della catena di polimerasi multiplex ancorata seguita dal sequenziamento di nuova generazione

Gene fusions frequently contribute to the oncogenic phenotype of many different types of cancer. Additionally, the presence of certain fusions in samples ...

Mapping the Structure-Function Relationships of Disordered Oncogenic Transcription Factors Using Transcriptomic Analysis

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these proteins contain an intrinsically disordered domain (IDD) fused with the DNA-binding domain (DBD) of another protein and orchestrate widespread transcriptional changes to promote malignancy. These fusions are often the sole recurring genomic aberration in the cancers they cause, making them attractive therapeutic targets. However, targeting oncogenic transcription factors requires a better understanding of the mechanistic role that low-complexity, IDDs play in their function. The N-terminal domain of EWSR1 is an IDD involved in a variety of oncogenic fusion transcription factors, including EWS/FLI, EWS/ATF, and EWS/WT1. Here, we use RNA-sequencing to investigate the structural features of the EWS domain important for transcriptional function of EWS/FLI in Ewing sarcoma. First shRNA-mediated depletion of the endogenous fusion from Ewing sarcoma cells paired with ectopic expression of a variety of EWS-mutant constructs is performed. Then RNA-sequencing is used to analyze the transcriptomes of cells expressing these constructs to characterize the functional deficits associated with mutations in the EWS domain. By integrating the transcriptomic analyses with previously published information about EWS/FLI DNA binding motifs, and genomic localization, as well as functional assays for transforming ability, we were able to identify structural features of EWS/FLI important for oncogenesis and define a novel set of EWS/FLI target genes critical for Ewing sarcoma. This paper demonstrates the use of RNA-sequencing as a method to map the structure-function relationship of the intrinsically disordered domain of oncogenic transcription factors.

Intrinsically disordered domains are important for oncogenic fusion transcription factor function. To therapeutically target these proteins, a more detailed understanding of the regulatory mechanisms employed by these domains is required. Here, we use transcriptomics to map important structural features of the intrinsically disordered EWS domain in Ewing sarcoma.

Mappatura delle relazioni struttura-funzione dei fattori di trascrizione oncogenica disordinati utilizzando l'analisi trascrittomica

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these ...

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

Una piattaforma di screening Luciferase basata multiplex per interrogare il cancro-associata trasduzione del segnale in cellule in coltura

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Medicina

Proximity Ligation Assay to Study Oncogene-Derived Transcription-Replication Conflicts

Both DNA replication and RNA transcription utilize genomic DNA as their template, necessitating spatial and temporal separation of these processes. Conflicts between the replication and transcription machinery, termed transcription-replication conflicts (TRCs), pose a considerable risk to genome stability, a critical factor in cancer development. While several factors regulating these collisions have been identified, pinpointing primary causes remains difficult due to limited tools for direct visualization and clear interpretation. In this study, we directly visualize TRCs using a proximity ligation assay (PLA), leveraging antibodies specific to PCNA and phosphorylated CTD of RNA polymerase II. This approach allows precise measurement of TRCs between replication and transcription processes mediated by RNA polymerase II. The method is further enhanced through DNA primers conjugated covalently to these antibodies, coupled with PCR amplification using fluorescent probes, providing a highly sensitive and specific means of detecting endogenous TRCs. Fluorescence microscopy enables the visualization of these conflicts, offering a powerful tool to study genome instability mechanisms associated with cancer. This technique addresses the gap in direct TRC visualization, allowing for a more comprehensive analysis and understanding of the underlying processes driving genome instability in cells.

Here, we present a sensitive and specific method to detect oncogene-induced transcription-replication conflicts (TRCs) using the proximity ligation assay (PLA). This approach leverages specific antibodies targeting PCNA and phospho-CTD RNAPII, enabling the assessment of TRC prevalence between RNA polymerase II transcription and DNA replication machinery.

Saggio di legatura di prossimità per studiare i conflitti di trascrizione-replicazione derivati da oncogeni

Both DNA replication and RNA transcription utilize genomic DNA as their template, necessitating spatial and temporal separation of these processes. ...

Oncogene Expression Analysis with Alterations in pH in a Pancreatic Ductal Cell Line

The fourth leading cause of cancer-related death, pancreatic ductal adenocarcinoma (PDAC), has a 12% five-year survival rate. This disease has a poor prognosis and is characterized by a rigid stromal microenvironment, which represents a tangible challenge in its treatment. Chronic pancreatitis patients have a 10-fold greater risk of developing PDAC; in these patients, the ductal pH decreases from pH 8.0 to pH 6.0 due to bicarbonate insufficiency and the inflammatory milieu. Our goal was to understand the role of the acidic environment observed in chronic pancreatitis on oncogene expression in a pancreatic ductal cell line.
Therefore, 80% confluent human pancreatic ductal epithelial cells were incubated at pH 6.0 to pH 7.2 for 6 h. Total RNA from the cells was processed to enrich the total mRNA in the samples. Gene expression was evaluated via next-generation sequencing (NGS) of biological replicates. RNA-seq analysis was carried out with the aid of an online tool, and the differentially expressed genes (FCs &#60; &#177; 2.0) were identified; there were 90, 148, and 109 upregulated genes and 20, 14, and 23 downregulated genes at pH 6.0, 6.5, and 7.0, respectively. Four oncogenes were upregulated at pH 6.0, seven were upregulated at pH 6.5, and seven were upregulated at pH 7.0. The common genes that were upregulated at pH 6.0, pH 6.5, and pH 7.0 were lymphocyte cell-specific protein-tyrosine kinase (LCK) [pH 6.0, FC: 2.93; pH 6.5, FC: 2.93; pH 7.0, FC: 3.32], FGR proto-oncogene, Src family tyrosine kinase (FGR) [pH 6.0, FC: 4.17; pH 6.5, FC: 5.25; pH 7.0, FC: 5.09], and ArfGAP With SH3 domain, ankyrin repeat, and PH domain 3 (ASAP3) [pH 6.0, FC: 2.37; pH 6.5, FC: 3.84; pH 7.0, FC: 2.51]. The acidic environment triggers the activation of proto-oncogenes, which may trigger tumor initiation in chronic pancreatitis.

This study describes a protocol for exploring the effects of altered pH on oncogene expression via RNA-seq analysis of a pancreatic ductal cell line.

Analisi dell'espressione di oncogeni con alterazioni del pH in una linea cellulare duttale pancreatica

The fourth leading cause of cancer-related death, pancreatic ductal adenocarcinoma (PDAC), has a 12% five-year survival rate. This disease has a poor ...

The Soft Agar Colony Formation Assay

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft agar colony formation assay is a well-established method for characterizing this capability in vitro and is considered to be one of the most stringent tests for malignant transformation in cells. This assay also allows for semi-quantitative evaluation of this capability in response to various treatment conditions. Here, we will demonstrate the soft agar colony formation assay using a murine lung carcinoma cell line, CMT167, to demonstrate the tumor suppressive effects of two members of the Wnt signaling pathway, Wnt7A and Frizzled-9 (Fzd-9). Concurrent overexpression of Wnt7a and Fzd-9 caused an inhibition of colony formation in CMT167 cells. This shows that expression of Wnt7a ligand and its Frizzled-9 receptor is sufficient to suppress tumor growth in a murine lung carcinoma model.

The soft agar colony formation assay is a method used to confirm cellular anchorage-independent growth in vitro. The goal of this protocol is to illustrate a stringent method for the detection of the tumorigenic potential of transformed cells and the tumor suppressive effects of proteins on transformed cells.

Il saggio Formazione morbida Agar Colony

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft ...