A high-content screening assay for small-molecule modulators of oncogene-induced senescence

J Biomol Screen. 2013 Oct;18(9):1054-61. doi: 10.1177/1087057113491827. Epub 2013 Jun 3.


Cellular senescence is a state of stable cell growth arrest. Activation of oncogenes such as RAS in mammalian cells typically triggers cellular senescence. Oncogene-induced senescence (OIS) is an important tumor suppression mechanism, and suppression of OIS contributes to cell transformation. Oncogenes trigger senescence through a multitude of incompletely understood downstream signaling events that frequently involve protein kinases. To identify target proteins required for RAS-induced senescence, we developed a small-molecule screen in primary human fibroblasts undergoing senescence induced by oncogenic RAS (H-Ras(G12V)). Using a high-content imaging system to monitor two hallmarks of senescence, senescence-associated β-galactosidase activity expression and inhibition of proliferation, we screened a library of known small-molecule kinase inhibitors for those that suppressed OIS. Identified compounds were subsequently validated and confirmed using a third marker of senescence, senescence-associated heterochromatin foci. In summary, we have established a novel high-content screening platform that may be useful for elucidating signaling pathways mediating OIS by targeting critical pathway components.

Keywords: cancer and cancer drugs; cell-based assays; high-content screening; kinases.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Biological Assay*
  • Cell Cycle Checkpoints / drug effects
  • Cell Proliferation / drug effects
  • Cellular Senescence / drug effects*
  • Cellular Senescence / genetics
  • Fibroblasts / cytology
  • Fibroblasts / drug effects*
  • Fibroblasts / metabolism
  • Gene Expression Regulation / drug effects
  • Genes, Reporter
  • Heterochromatin / drug effects
  • Heterochromatin / metabolism
  • Humans
  • Oncogene Protein p21(ras) / genetics
  • Oncogene Protein p21(ras) / metabolism
  • Primary Cell Culture
  • Protein Kinase Inhibitors / chemistry
  • Protein Kinase Inhibitors / pharmacology*
  • Signal Transduction / drug effects*
  • Small Molecule Libraries / chemistry
  • Small Molecule Libraries / pharmacology*
  • beta-Galactosidase / genetics
  • beta-Galactosidase / metabolism


  • Heterochromatin
  • Protein Kinase Inhibitors
  • Small Molecule Libraries
  • beta-Galactosidase
  • Oncogene Protein p21(ras)