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BH3 Profiling: A Functional Assay to Measure Apoptotic Priming and Dependencies

  • Cameron Fraser
  • Jeremy Ryan
  • Kristopher Sarosiek
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1877)

Abstract

Apoptosis (programmed cell death) is activated by a wide variety of cellular stresses or insults and is vital for proper mammalian development as well as the maintenance of organismal homeostasis. The apoptosis pathway is also engaged by many common types of anticancer therapies and ionizing radiation, which contributes to the regressions of tumors or the toxic side effects of treatment. Due to the importance of maintaining healthy cell survival or the efficient clearance of cancer cells, the BH3 profiling assay was developed to functionally measure the state of the apoptosis pathway in any given cells. This assay involves the exposure of cellular mitochondria, where the BCL-2 family of proteins resides and controls the commitment to apoptosis, to proapoptotic BH3 peptides that mimic the activity of endogenous proapoptotic proteins. By using either activator or sensitizer peptides, the level of mitochondrial apoptotic priming (proximity to the threshold at which a cell commits to cell death) or dependence on prosurvival BCL-2 family proteins can be determined. Described here are two methods of BH3 profiling that can enable a user to make these functional measurements, which can be useful for predicting cellular responses to proapoptotic stressors or therapeutics (BH3 mimetics) that inhibit the activity of prosurvival proteins.

Key words

Mitochondrial apoptotic priming MOMP BCL-2 family Chemotherapeutics 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Cameron Fraser
    • 1
    • 2
  • Jeremy Ryan
    • 3
  • Kristopher Sarosiek
    • 1
    • 2
  1. 1.John B. Little Center for Radiation Sciences, Harvard T.H. Chan School of Public HealthBostonUSA
  2. 2.Department of Environmental HealthHarvard T.H. Chan School of Public HealthBostonUSA
  3. 3.Department of Medical OncologyDana-Farber Cancer InstituteBostonUSA

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