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

Summary

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

Abstract

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.

Introduction

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 form....

Protocol

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.

1. Prepare transfection-grade plasmid DNA from competent cells using the QIAGEN plasmid midi kit.
2. Measure DNA concentration using a NanoDrop2000 spectrophotometer.

2. Retrovirus Production

The Plat-E packaging cell line wi....

Results

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 family^13-15, and it has been reported to be involved in carcinogenesis^16,17. 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.......

Discussion

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 “cooperation” 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.......

Materials

List of materials used in this article

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