Driving gradual endogenous c-myc overexpression by flow-sorting: intracellular signaling and tumor cell phenotype correlate with oncogene expression.
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.
Knudsen
Kasper Jermiin
KJ
Institute for Micro and Nanotechnology, Technical University of Denmark, Lyngby, Denmark.
Nelander Holm
Gitte-Mai
GM
Krabbe
Jonas S
JS
Listov-Saabye
Nicolai
N
Kiehr
Benedicte
B
Dufva
Martin
M
Svendsen
Jette E
JE
Oleksiewicz
Martin B
MB
D000230:Adenocarcinoma / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D000818:Animals; D002448:Cell Adhesion; D045744:Cell Line, Tumor; D002469:Cell Separation; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D006657:Histones / Q000378:metabolism; D007328:Insulin / Q000494:pharmacology; D015674:Mammary Neoplasms, Animal / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D010641:Phenotype; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism*; D051381:Rats; D017526:Receptor, IGF Type 1 / Q000378:metabolism; D015398:Signal Transduction
D006657:Histones; D007328:Insulin; D016271:Proto-Oncogene Proteins c-myc; D019869:Phosphatidylinositol 3-Kinases; D017526:Receptor, IGF Type 1; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3