Assessment of HDACi-Induced Protein Cleavage by Caspases

Methods Mol Biol. 2017:1510:11-22. doi: 10.1007/978-1-4939-6527-4_2.

Abstract

Aberrant histone deacetylase (HDAC) activity often correlates with neoplastic transformation and inhibition of HDACs by small molecules has emerged as a promising strategy to treat hematological malignancies in particular. Treatment with HDAC inhibitors (HDACis) often prompts tumor cells to undergo apoptosis, thereby causing a caspase-dependent cleavage of target proteins. An unexpectedly large number of proteins are in vivo caspase substrates and defining caspase-mediated substrate specificity is a major challenge. In this chapter we demonstrate that the hematopoietic transcription factor PU.1 becomes cleaved after treatment of acute myeloid leukemia (AML) cells with the HDACis LBH589 (panobinostat) or MS-275 (entinostat). To define caspase specificity for PU.1, an in vitro caspase assay including caspases 1-10 with in vitro-translated PU.1 is described in detail.

Keywords: Caspase-8; HDACi; In vitro translation; LBH589; MS-275; PU.1.

MeSH terms

  • Antineoplastic Agents / pharmacology*
  • Benzamides / pharmacology
  • Blotting, Western / methods
  • Caspase 8 / genetics*
  • Caspase 8 / metabolism
  • Electrophoresis, Polyacrylamide Gel / methods
  • Enzyme Activation
  • Gene Expression Regulation, Neoplastic*
  • HEK293 Cells
  • HL-60 Cells
  • Histone Deacetylase Inhibitors / pharmacology*
  • Histone Deacetylases / genetics
  • Histone Deacetylases / metabolism
  • Humans
  • Hydroxamic Acids / pharmacology
  • Indoles / pharmacology
  • K562 Cells
  • Panobinostat
  • Proteolysis
  • Proto-Oncogene Proteins / genetics*
  • Proto-Oncogene Proteins / metabolism
  • Pyridines / pharmacology
  • Substrate Specificity
  • Trans-Activators / genetics*
  • Trans-Activators / metabolism
  • Transfection

Substances

  • Antineoplastic Agents
  • Benzamides
  • Histone Deacetylase Inhibitors
  • Hydroxamic Acids
  • Indoles
  • Proto-Oncogene Proteins
  • Pyridines
  • Trans-Activators
  • proto-oncogene protein Spi-1
  • entinostat
  • Panobinostat
  • CASP8 protein, human
  • Caspase 8
  • Histone Deacetylases