Name: focus formation: a cell-based assay to determine the oncogenic potential of a gene
Uploaded: 2014-12-31T15:00:00.000+00:00
Duration: 8 min 18 s
Description: Watch this Scientific Journal Video about Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene at JoVE.com

This work was supported by a grant from the NIH Director&#39;s New Innovator Award Program (E.D.). A. A. was supported in part by undergraduate awards from the National Cancer Institute and the National Science Foundation.

1. Making the Viral Vectors
The coding sequences for the gene of interest, as well as the positive and negative controls, are inserted into pBABEpuro by traditional cloning methods (PCR amplification, restriction digestion and ligation). There are four restriction sites on the vector where the DNA can be inserted: BamHI, SnaBI, EcoRI and SalI.
<ol>
	<li>Prepare transfection-grade plasmid DNA from competent cells using the QIAGEN plasmid midi kit.</li>
	<li>Measure DNA concentration using a NanoDrop2000 spectrophotometer.</li>
</ol>
2. Retrovirus Production
The Plat-E packaging cell line will be used to produce ecotropic retrovirus that will deliver the cDNA of interest to NIH 3T3 cells.
<ol>
	<li>Establishing Plat-E Cultures from Frozen Cells
	<ol>
		<li>Quickly thaw Plat-E cells in a 37 &#176;C water bath and transfer to a 15 ml conical tube. Slowly add 9 ml of Plat-E medium (Dulbecco&#8217;s Modified Eagle Medium (DMEM) + 10% Fetal Bovine Serum (FBS) + Penicillin and Streptomycin).</li>
		<li>Centrifuge the tube at 180 x g for 5 min and discard the supernatant.</li>
		<li>Re-suspend the cells with 10 ml of Plat-E medium and transfer to a 10 cm culture dish.</li>
		<li>Incubate the cells in a 37 &#176;C, 5% CO2 incubator until they are 80-90% confluent (about 2 days).</li>
	</ol>
	</li>
	<li>Cell Splitting
	<ol>
		<li>Aspirate the medium and wash cells once with PBS.</li>
		<li>Add 2 ml of 0.05% trypsin-EDTA and incubate for 1 min at RT.</li>
		<li>Detach the cells by finger tapping, add 10 ml of Plat-E medium, and transfer the cell suspension to a 15 ml tube.</li>
		<li>Centrifuge the tube at 180 x g for 5 min and discard the supernatant.</li>
		<li>Resuspend the cells with 10 ml of Plat-E medium and seed them in new dishes at 1:4-1:6 dilutions.</li>
	</ol>
	</li>
	<li>Plat-E Seeding and Transfection
	<ol>
		<li>Seed 2 x 106 cells per 10 cm culture dish using Plat-E medium without any antibiotics.</li>
		<li>Incubate the cells O/N in a 37 &#176;C, 5% CO2 incubator.</li>
		<li>Next day, transfer 300 &#956;l of Opti-MEM into 1.5 ml microfuge tube.</li>
		<li>Add 27 &#956;l of polyethylenimine (PEI), a cost-effective transfection reagent12, to the prepared tube with Opti-MEM. Mix gently by finger tapping and incubate for 5 min at RT.</li>
		<li>Add 9 &#956;g of transfection-grade plasmid DNA into the Opti-MEM/PEI tube, mix gently by vortexing, and incubate for 15 min at RT.</li>
		<li>Add the DNA/PEI complex drop-wise into the Plat-E dish, and incubate O/N&#160;at 37 &#176;C, 5% CO2.</li>
		<li>Next day, aspirate the medium containing the transfection reagent and add 10 ml of fresh Plat-E medium (without antibiotics).</li>
		<li>Return the cells to the incubator.</li>
	</ol>
	</li>
</ol>
3. NIH 3T3 Cells and Infection
<ol>
	<li>Establishing NIH 3T3 Cultures and Seeding
	<ol>
		<li>Quickly thaw NIH 3T3 cells in a 37 &#176;C water bath and transfer to a 15 ml tube. Slowly add 9 ml of NIH 3T3 medium (DMEM + 10% FBS).</li>
		<li>Centrifuge the tube at 180 x g for 5 min and discard the supernatant.</li>
		<li>Resuspend the cells with 10 ml of NIH 3T3 medium and transfer to a 10 cm culture dish.</li>
		<li>Incubate the cells in a 37 &#176;C, 5% CO2 incubator. It is very important that NIH 3T3 cells are always kept underconfluent (50-60%), as the frequency of spontaneous transformation (and therefore basal levels of foci formation) increases once a culture reaches confluency.</li>
		<li>Seed 3 x 105 cells per 10 cm dish and incubate O/N for infection the next day.</li>
	</ol>
	</li>
	<li>Infection
	<ol>
		<li>Harvest the retroviral supernatant from the Plat-E dish by using a 10 ml disposable syringe, filtering it through a 0.45 &#956;m pore size nylon membrane filter, and transferring it into a 15 ml tube.</li>
		<li>Add 10 ml of fresh Plat-E medium to the cells (without antibiotics), and return them to the incubator.</li>
		<li>Aspirate the medium from the NIH 3T3 cell dish to be infected, and add 5 ml of regular NIH 3T3 medium and 5 ml of virus-containing filtered supernatant.</li>
		<li>Add polybrene to the dish at a concentration of 6 &#956;g/ml.</li>
		<li>Incubate the cells O/N at 37 &#176;C, 5% CO2.</li>
		<li>Next day, repeat steps 3.2.1 &#8211; 3.2.5 for a second round of infection.</li>
		<li>Replace the virus-containing medium with regular NIH 3T3 medium.</li>
		<li>Allow the NIH 3T3 cells to grow for 2-3 weeks, replacing the medium as necessary. Check expression of the protein of interest by conventional protein electrophoresis and immunoblot on whole cell lysates from replica plates.</li>
	</ol>
	</li>
</ol>
4. Staining and Quantification
<ol>
	<li>Crystal Violet Staining
	<ol>
		<li>Aspirate the NIH 3T3 medium and place the dishes on ice.</li>
		<li>Wash the dishes twice with ice-cold PBS.</li>
		<li>Fix the cells with ice-cold methanol for 10 min.</li>
		<li>Remove the dishes from ice, aspirate the methanol, and add 3 ml of 0.5% crystal violet solution made in 25% methanol (RT).</li>
		<li>Incubate the dishes for 5 min at RT.</li>
		<li>Aspirate the crystal violet solution and carefully rinse the dish with Milli-Q H2O until no color comes off in the rinse.</li>
		<li>Allow the dishes to dry O/N&#160;on a benchtop (uncovered).</li>
	</ol>
	</li>
	<li>Foci Quantification
	<ol>
		<li>Once the dishes are dry, use a ruler to measure the darkly colored collections of cells on the plate. Only count those that are greater than 5 mm in diameter as &#8220;foci.&#8221; Record the number of foci and calculate averages and the significance compared to the controls.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

The FFA provides a quick and easy method to evaluate malignant transformation in vitro. It is amenable to screening of a relatively large number of candidate genes, and its modest technical requirements make it cost-effective. Furthermore, two or more genes can be coexpressed (sometimes referred to as a &#8220;cooperation&#8221; assay) to evaluate the tumorigenic potential of the combination. The advantages of this assay rely on its straightforward technique, its ease of quantification and its relatively short t...

Tumor cells can be distinguished from their normal counterparts by a wide range of alterations, from patterns of gene expression to epigenomics to morphological and proliferative changes. Among the latter, reduced dependence on serum, loss of contact (density) inhibition, the acquisition of anchorage-independent proliferation and, ultimately, the ability to form tumors when injected into animals are useful, measurable indicators of malignant transformation3. Several in vitro and in vivo assays have been developed for cellular transformation. In vitro assays aim at identifying and measuring changes in culture morphology (focus formation assay), culture dynamics (growth rate, saturation density) and growth factor (growth in reduced serum) or anchorage (growth in soft agar) requirements. The gold standard for determining the malignant nature of a cell type remains tumor formation (xenografts) in experimental animals. However, the cost and length of in vivo studies do not always make them justifiable as a first validation step or screening of candidate oncogenes. Although no in vitro assay provides a definite assessment of the oncogenic potential of a gene, they do provide insight into oncogenic potential that may narrow down future in vivo studies. One of the most widely used systems for evaluating oncogenic potential in vitro is the Focus Formation Assay2. This approach relies on the use of NIH 3T3 mouse fibroblast, a non-transformed cell line that shows strong contact inhibition. Overexpression of an oncogene results in loss of density-dependent growth; transformed cells can then grow in multiple layers, forming &#8220;foci&#8221;, easily visualized against the background monolayer of non-transformed cells. The Focus Formation Assay, then, measures the ability of a candidate oncogene to induce malignant transformation, as evidenced by the loss of contact inhibition as a measurable phenotype. The FFA has been used to evaluate transformation by overexpression of protein kinases (e.g., Src4, BRAF5), transcription factors (e.g., N-myc6), G-protein-coupled receptors (e.g., P2RY87) and GTPases (e.g., Ras1), among others. The relative ease of this assay makes it a good choice that will provide a quick and visually clear answer to whether overexpression of the gene is sufficient to transform NIH 3T3 mouse fibroblast cells in vitro.
The FFA described in this protocol uses the Plat-E packaging cell line8, which provides viral packaging proteins, and the retroviral vector pBABEpuro9 (Addgene plasmid 1764) to produce retrovirus. After transfection with the pBABEpuro construct containing the gene of interest, the Plat-E cell line will produce ecotropic retrovirus that can be used to infect NIH 3T3 cells. This viral method of gene delivery is more efficient than traditional chemical transfection methods and it offers a way to sustainably express the gene10. Once incorporated into the genome of the NIH 3T3 cells, overexpression of the gene of interest is driven by the viral long terminal repeats (LTR) promoter11. This constant expression can be used to determine whether the gene of interest has oncogenic activity, as measured by the formation of foci, on the NIH 3T3 cells.

<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 is a basic helix-loop-helix leucine zipper (bHLHZ) transcription factor that is a member of the MYC/MAX/MAD network. It is an atypical member of the MAD family13-15, and it has been reported to be involved in carcinogenesis16,17. When compared to pBABEpuro (negative control) and MYC (positive control), the NIH 3T3 dishes where MXD3 was overexpressed had significantly less foci (Figure 1A). The data in Figure 1B were collected from multiple experiments to determ...

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

The Focus Formation Assay provides a straightforward method to assess the transforming potential of a candidate oncogene.

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

focus-formation-cell-based-assay-to-determine-oncogenic-potential

Universidad de Cartagena UDEC

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JoVE Journal

Medicine

27.8K Views.  University of California, Davis. The Focus Formation Assay provides a straightforward method to assess the transforming potential of a candidate oncogene.

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.

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

Cancer Research

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.

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

Immunology and Infection

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.

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

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.

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

The Use of Mouse Mammary Tumor Cells in an In Vitro Invasion Assay as a Measure of Oncogenic Cell Behavior

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a porous membrane in a process analogous to the initial steps of cancer cell metastasis. The tested cells can be altered for the gene expression or treated with inhibitors to test for changes in the invasion potential. This experiment tests the aggressive phenotype of the mouse mammary tumor cells to discover and characterize the potential oncogenes that promote cell invasion. This technique, however, can be versatile and adapted to many different applications. The experiment itself can be done in one day and the results are acquired by light microscopy in less than a day. The results include counts of the number of invading cells for comparison and analysis. The in vitro invasion assay is a rapid, inexpensive, and clear-cut method for determining cell behavior in a culture that can be used as an initial assessment before more involved in vivo assays.

The in vitro cell invasion assay is used to measure the potential of cancer metastasis by quantifying the cellular potential for invasion and migration using cell culture inserts containing protein matrix. Cells are challenged to migrate through the protein matrix and a porous membrane, towards a chemoattractant, and then quantified by light microscopy.

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a ...

Genetics

Tumorsphere Derivation and Treatment from Primary Tumor Cells Isolated from Mouse Rhabdomyosarcomas

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Although significant efforts have enabled the identification of common mutations associated with RMS and allowed discrimination of different RMS subtypes, major challenges still exist for the development of novel treatments to further improve prognosis. Although identified by the expression of myogenic markers, there is still significant controversy over whether RMS has myogenic or non-myogenic origins, as the cell of origin is still poorly understood. In the present study, a reliable method is provided for the tumorsphere assay for mouse RMS. The assay is based on functional properties of tumor cells and allows the identification of rare populations in the tumor with tumorigenic functions. Also described are procedures for testing recombinant proteins, integrating transfection protocols with the tumorsphere assay, and evaluating candidate genes involved in tumor development and growth. Described further is a procedure for allograft transplantation of tumorspheres into recipient mice to validate tumorigenic function in vivo. Overall, the described method allows reliable identification and testing of rare RMS tumorigenic populations that can be applied to RMS arising in different contexts. Finally, the protocol can be utilized as a platform for drug screening and future development of therapeutics.

This protocol describes a reproducible method for isolation of mouse rhabdomyosarcoma primary cells, tumorsphere formation and treatment, and allograft transplantation starting from tumorspheres cultures.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Although significant efforts have enabled the identification of common ...

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.

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.

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

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.

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

Biology

Methyl Cellulose-Based CFU Assay: A Method to Determine the Effect of Anti-cancer Compounds on Colony Formation in Leukemic Cells

Source: Kesarwani, M., et al. <a href="https://jove.unicartagenaproxy.elogim.com/t/58194">Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence.</a> J. Vis. Exp. (2019).
This video describes the protocol for methyl cellulose-based colony formation unit or CFU assay, which helps identify and count the number of colonies formed by leukemic cells in response to the treatment with anticancer compounds.

Source: Kesarwani, M., et al. Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence. J. Vis. Exp. (2019).
This video describes the ...