title: Quantitative assessment of Ras over-expression via shotgun deployment of vectors utilizing synthetic promoters.
abstract: 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.
authors: Department of Chemical Engineering, Stanford University, 381 N-S Axis, Rm 113, Stanford, CA 94305, USA.
authors: Joshua P
authors: JP
authors: Ferreira
authors: Ingrid E B
authors: IE
authors: Lawhorn
authors: Ryan W S
authors: RW
authors: Peacock
authors: Clifford L
authors: CL
authors: Wang
mesh_terms: D001549:Benzamides; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D011905:Genes, ras; D006801:Humans; D000068877:Imatinib Mesylate; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism*; D010879:Piperazines / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011743:Pyrimidines / Q000494:pharmacology*; D015398:Signal Transduction; D018631:ras Proteins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D001549:Benzamides; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D000068877:Imatinib Mesylate; D020929:Mitogen-Activated Protein Kinase Kinases; D018631:ras Proteins