Single-cell analysis challenges the connection between autophagy and senescence induced by DNA damage

Autophagy. 2015;11(7):1099-113. doi: 10.1080/15548627.2015.1009795.

Abstract

Autophagy and senescence have been described as central features of cell biology, but the interplay between these mechanisms remains obscure. Using a therapeutically relevant model of DNA damage-induced senescence in human glioma cells, we demonstrated that acute treatment with temozolomide induces DNA damage, a transitory activation of PRKAA/AMPK-ULK1 and MAPK14/p38 and the sustained inhibition of AKT-MTOR. This produced a transient induction of autophagy, which was followed by senescence. However, at the single cell level, this coordinated transition was not observed, and autophagy and senescence were triggered in a very heterogeneous manner. Indeed, at a population level, autophagy was highly negatively correlated with senescence markers, while in single cells this correlation did not exist. The inhibition of autophagy triggered apoptosis and decreased senescence, while its activation increased temozolomide-induced senescence, showing that DNA damage-induced autophagy acts by suppressing apoptosis.

Keywords: 3MA, 3-methyladenine; AMP-activated; AO, acridine orange; BafA1, bafilomycin A1; CDKN1A/p21, cyclin-dependent kinase inhibitor 1A (p21 Cip1); CPD, cumulative population doubling; DDR, DNA damage response; DFM, drug-free medium; DNA damage; H2AFX, H2A histone family; MAP1LC3A/LC3, microtubule-associated protein 1 light chain 3 α; MTOR, mechanistic target of rapamycin; MTORC1, MTOR complex 1; NA, nuclear area; NMA, nuclear morphometric analysis; PRKAA/AMPKα, protein kinase; RAPA, rapamycin; RPTOR/RAPTOR, regulatory-associated protein of MTOR; SA-β-gal, senescence associated β-galactosidase assay; SQSTM1/p62, sequestosome 1; TMZ, temozolomide; autophagy; cP1-4, cellular population 1 to 4; complex 1; member X; nP1–5, nuclear population 1 to 5; senescence; single cell; temozolomide.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • AMP-Activated Protein Kinases / metabolism
  • Apoptosis / drug effects
  • Autophagy* / drug effects
  • Autophagy-Related Protein-1 Homolog
  • Cell Cycle Checkpoints / drug effects
  • Cell Line, Tumor
  • Cellular Senescence* / drug effects
  • DNA Damage*
  • Dacarbazine / analogs & derivatives
  • Dacarbazine / pharmacology
  • Enzyme Activation / drug effects
  • Glioma / enzymology
  • Glioma / pathology
  • Green Fluorescent Proteins / metabolism
  • Humans
  • Intracellular Signaling Peptides and Proteins / metabolism
  • Models, Biological
  • Phenotype
  • Protein-Serine-Threonine Kinases / metabolism
  • Single-Cell Analysis / methods*
  • Sirolimus / pharmacology
  • TOR Serine-Threonine Kinases / metabolism
  • Temozolomide
  • Time Factors
  • p38 Mitogen-Activated Protein Kinases / metabolism

Substances

  • Intracellular Signaling Peptides and Proteins
  • Green Fluorescent Proteins
  • Dacarbazine
  • TOR Serine-Threonine Kinases
  • Autophagy-Related Protein-1 Homolog
  • Protein-Serine-Threonine Kinases
  • ULK1 protein, human
  • p38 Mitogen-Activated Protein Kinases
  • AMP-Activated Protein Kinases
  • Sirolimus
  • Temozolomide