BH3 profiling--measuring integrated function of the mitochondrial apoptotic pathway to predict cell fate decisions

Abstract

Apoptosis is a form of programmed cell death that is controlled at the mitochondrion by the BCL-2 family of proteins. While much has been learned about the structure and function of these proteins over the past two decades, the important goal of predicting cell fate decisions in response to toxic stimuli is largely unrealized. BH3 profiling is a functional approach that can be used to predict cellular responses to stimuli based on measuring the response of mitochondria to perturbation by a panel of BH3 domain peptides. BH3 profiling has proven useful in identifying and understanding cellular dependence on individual anti-apoptotic proteins like BCL-2 or MCL-1. Consequently, it can also be used to predict cellular response to chemotherapy agents such as ABT-737 that target these individual proteins.

Figure 1. Model of apoptotic control by BCL-2 family proteins

Cellular damage induces upregulation of activator and/or sensitizer proteins. BAX and BAK oligomerization is then induced by direct binding of activators like BID and BIM leading to MOMP then cytochrome c release and subsequent cell death. Anti-apoptotic proteins inhibit apoptosis by binding and sequestering activators or activated BAX or BAK. Sensitizers can bind to anti-apoptotic proteins and displace pre-bound activators, or previously activated BAX or BAK, inducing BAX/BAK oligomerization and mitochondrial permeabilization. (adapted from Certo et al. [15]).

Figure 2. The binding code

Summary of the selective binding between BH3-only peptides and different antiapoptotic BCL-2 family members. + indicates moderate binding, ++ tight binding, and – no binding interaction based on fluorescent polarization binding studies (adapted from Certo et al. [15]).

Figure 3. BH3 Profiling Assay Protocols

Flow charts representing the various steps for BH3 profiling using cytochrome c ELISA (A), JC-1 in a plate reader format(B), and JC-1 with flow cytometry (C) (adapted from Ryan et al [33]).

Figure 4. BH3 profiling using isolated mitochondria and whole cell assays produce comparable results

BCL-2 1863, MCL-1 1780 and MDA-MB-231 cell lines were used to either isolate mitochondria via a heavy membrane preparation and profiled with 100μM peptides either using a cytochrome c ELISA (HM-CC) (A) or whole cells used in the JC-1 (WC-JC-1) profiling assay (B). Two Upper panels in A represent data from a single experiment while all the rest triplicates; means shown with bars for SD (adapted from Ryan et al [33]).