title: A high-content screening assay for small-molecule modulators of oncogene-induced senescence.
abstract: 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.
authors.affiliation: 1Gene Expression and Regulation Program, The Wistar Institute, Philadelphia, PA, USA.
authors.forename: Benjamin G
authors.initials: BG
authors.lastname: Bitler
authors.forename: Lauren S
authors.initials: LS
authors.lastname: Fink
authors.forename: Zhi
authors.initials: Z
authors.lastname: Wei
authors.forename: Jeffrey R
authors.initials: JR
authors.lastname: Peterson
authors.forename: Rugang
authors.initials: R
authors.lastname: Zhang
mesh_terms: D001681:Biological Assay; D059447:Cell Cycle Checkpoints / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D016922:Cellular Senescence / Q000187:drug effects* / Q000235:genetics; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D006570:Heterochromatin / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D015689:Oncogene Protein p21(ras) / Q000235:genetics / Q000378:metabolism; D061251:Primary Cell Culture; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects*; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
chemical_list: D006570:Heterochromatin; D047428:Protein Kinase Inhibitors; D054852:Small Molecule Libraries; D001616:beta-Galactosidase; D015689:Oncogene Protein p21(ras)
keywords: cancer and cancer drugs; cell-based assays; high-content screening; kinases