Journal article
Driving gradual endogenous c-myc overexpression by flow-sorting: intracellular signaling and tumor cell phenotype correlate with oncogene expression
Department of Micro- and Nanotechnology, Technical University of Denmark1
Fluidic Array Systems and Technology Group, Biomedical Micro Systems Section, Department of Micro- and Nanotechnology, Technical University of Denmark2
Biomedical Micro Systems Section, Department of Micro- and Nanotechnology, Technical University of Denmark3
Insulin-exposed rat mammary cancer cells were flow sorted based on a c-myc reporter plasmid encoding a destabilized green fluorescent protein. Sorted cells exhibited gradual increases in c-myc levels. Cells overexpressing c-myc by only 10% exhibited phenotypic changes attributable to c-myc overexpression, such as cell cycle disturbances, increased cell size, and overexpression of the S6 ribosomal protein.
Cells overexpressing c-myc by 70% exhibited additional phenotypic changes typical of c-myc overexpression, such as increased histone H3 phosphorylation, and reduced adherence. Sorted cells also exhibited overexpression of the IGF-1R, and slightly elevated expression of the IR. Increased susceptibility to the mitogenic effect of insulin was seen in a small proportion of the sorted cells, and insulin was more effective in activating the p44/42 MAPK pathway, but not the PI3K pathway, in the sorted cells than in the nonsorted cell population.
To our knowledge, this is the first in vitro system allowing functional coupling between mitogenic signaling by a well-defined growth factor and gradual overexpression of the normal, endogenous c-myc gene. Thus, our flow-sorting approach provides an alternative modeling of the receptor-mediated carcinogenic process, compared to the currently used approaches of recombinant constitutive or conditional overexpression of oncogenic transmembrane receptor tyrosine kinases or oncogenic transcription factors.
Language: | English |
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Publisher: | Springer-Verlag |
Year: | 2009 |
Pages: | 1061-1074 |
ISSN: | 14320738 and 03405761 |
Types: | Journal article |
DOI: | 10.1007/s00204-009-0463-6 |
ORCIDs: | Dufva, Martin |
Fluorescence-activated cell sorting Green fluorescent protein Insulin mitogenicity Molecular toxicology Preclinical carcinogenicity safety assessment SDG 3 - Good Health and Well-being c-myc
Adenocarcinoma Animals Biomedicine Biomedicine general Cell Adhesion Cell Line, Tumor Cell Separation Environmental Health Female Flow Cytometry Histones Insulin Mammary Neoplasms, Animal Mitogen-Activated Protein Kinase 1 Mitogen-Activated Protein Kinase 3 Occupational Medicine/Industrial Medicine Pharmacology/Toxicology Phenotype Phosphatidylinositol 3-Kinases Phosphorylation Proto-Oncogene Proteins c-myc Rats Receptor, IGF Type 1 Signal Transduction