Quantitative assessment of Ras over-expression via shotgun deployment of vectors utilizing synthetic promoters

• Published in: Integrative biology (Cambridge) 2012-01, Vol.4 (1), p.108-114
• Main Authors: Ferreira, Joshua P, Lawhorn, Ingrid E B, Peacock, Ryan W S, Wang, Clifford L
• Format: Article
• Language: English
• Subjects: Benzamides; Cell Line; Cell Proliferation - drug effects; Dose-Response Relationship, Drug; Genes, ras; Humans; Imatinib Mesylate; Mitogen-Activated Protein Kinase Kinases - metabolism; Piperazines - pharmacology; Promoter Regions, Genetic; Protein Kinase Inhibitors - pharmacology; Pyrimidines - pharmacology; ras Proteins - biosynthesis; ras Proteins - genetics; Signal Transduction
• ISSN: 1757-9708; 1757-9694; 1757-9708
• DOI: 10.1039/c1ib00082a

We sought to characterize and compare wild-type and oncogenic Ras over-expression. Because different levels of Ras over-expression can have different effects on cell phenotype, it was important to evaluate a wide range of expression. Different expression levels were achieved by using retroviral vectors equipped with different strength promoters. Cells were "shotgun" transduced with a mixture of these vectors to generate heterogeneous populations exhibiting a range of expression levels. We used flow cytometry to analyze the populations and generate high-resolution, nearly continuous Ras dose-response curves. These efforts revealed that a single-copy level of oncogenic Ras generated maximal imatinib resistance and activated MAPK pathway signaling as effectively as six-fold amplification of wild-type Ras. Although further increased expression lead to even greater signal transduction, this increased expression had minimal or decreasing effects on the proliferation rate. In addition, this study introduces a general method to quantify genetic dose-response relationships and identify gene expression ranges that produce an optimized phenotypic response.