Elsevier

Methods

Volume 61, Issue 2, 1 June 2013, Pages 156-164
Methods

BH3 profiling in whole cells by fluorimeter or FACS

https://doi.org/10.1016/j.ymeth.2013.04.006Get rights and content

Highlights

  • Functional assay reports mitochondrial response without knowledge of protein levels.

  • Plate reader assay is suitable for large sample numbers for homogenous samples.

  • FACS assay can determine priming in sub-populations from heterogeneous samples.

  • Suitable for any viable single cell suspension including primary samples.

Abstract

Rapid analysis of a cell’s propensity to undergo apoptosis through the mitochondrial pathway is hindered by the complex network of interactions between more than fifteen known members of the BCL2 family that govern the decision to undergo mitochondrial apoptosis, and measurement of protein levels alone fails to account for critical interactions between the proteins. To address this issue, we have developed two functional assays for same-day analysis of cell lines or primary tissue samples. Using defined inputs in the form of peptides derived primarily from the BH3 domains of pro-apoptotic members of the BCL2 family, we invoke a response in the mitochondria in the form of mitochondrial outer membrane permeabilization measured indirectly using potential sensitive dyes. BH3 profiling can be applied to any viable single cell suspension and provides a response from the sum total of all known and unknown interactions within the BCL2 family for each stimulus, and the pattern of response can provide both a cell’s propensity towards mitochondrial apoptosis, or ‘priming’, as well as indicate dependencies on specific anti-apoptotic proteins. Described here are optimized conditions for both plate-based and FACS-based BH3 profiling for homogeneous and heterogeneous samples.

Introduction

The mitochondrial pathway of apoptosis stands as a critical component in the development and maintenance of complex, multicellular organisms [1]. This pathway furthermore mediates the cell fate response to many toxic insults, including those therapeutically introduced into cancer patients by oncologists. The BCL-2 family of proteins controls the mitochondrial outer membrane permeabilization (MOMP) that is the key step in commitment to cell death by this pathway. There are many situations, both investigational and clinical, in which it would be useful to predict whether or not a given cell will undergo apoptosis in response to a particular insult. One might presume that comprehensive knowledge of the BCL-2 family proteins in a cell would provide the information required to provide such a prediction. However, the BCL-2 family proteins which regulate the mitochondrial apoptotic pathway are numerous and have a complex pattern of interaction [2]. Identifying all the interactions, expression levels, and post-translational modifications of the more than fifteen known members [3] of the family is by itself a feat beyond our accomplishing at present. Moreover, it is not clear that a model can be developed to receive these inputs that can be generalized among different cell types and insults.

Apoptosis may be thought of like a car, a very complex machine with a number of elements and interactions that any one of us could potentially isolate and examine. However, in practice most of us have a very incomplete understanding of it works in any detail. However, one does not need to measure the interaction of every gear to know that a fixed input from the accelerator will result in a proportional output from the tires. For most of us, this association of accelerator with motion is all we ever need to comprehend to operate an automobile. Similarly, while we may never understand the interactions of all of the BCL-2 family proteins simultaneously, the study of simple inputs and outputs of the mitochondrial apoptosis pathway we can nonetheless derive useful, actionable information.

The BCL2 family can be divided into three groups: multi-domain anti-apoptitic, multi-domain pro-apoptotic, and BH3-only pro-apoptotics. Multi-domain pro-apoptotics BAX and BAK are responsible forming the pores that cause MOMP following an activating conformational change and insertion into the mitochondrial outer membrane. The multi-domain antiapoptotic proteins all share a hydrophobic groove where the BH3-helix of the BH3 domain can bind and be inhibited or bind and displace another resident BCL2 protein [4]. It is from the BH3 helix of the BH3-only proteins that the peptides used here are derived and hence the name BH3 profiling.

Peptides used in BH3 profiling (Table 1) are all 20–25 mers containing the conserved LXXXXD motif, and can be divided into two categories: activators and sensitizers. The major difference in behavior between these two classes is the ability to directly induce the activating conformational change in BAX and BAK that leads to MOMP. BIM and BID peptides alone possess this ability while, regardless of their protein of origin, BAD, NOXAA, PUMA, BMF, and HRK derived peptides used in profiling cannot induce this change even at concentrations as high as 100 μM and are thus designated as sensitizer peptides [5].

BH3 profiling is a functional assay that uses a defined input in the form of small molecules or peptides, most often derived from the BH3 domain of known Bcl-2 family members, to provoke a response from mitochondria. The responses measured are typically surrogates of MOMP, namely release of cytochrome c or the collapse of the transmembrane mitochondrial potential (ΔΨm) [2], [6], [7], [8], [9]. It is capable of taking the sum total of all modifications into account during its measurement without needing to directly probe for each modification or protein level. Furthermore, the assay is swift enough to be completed in a matter of hours from the time of sample arrival. BH3 profiling is a rapid and simple method of probing the functional competence of the intrinsic apoptotic pathway and a cell’s propensity to undergo apoptosis.

Described here are the methods suitable for performing BH3 profiling on both homogeneous and heterogeneous cell populations from cell lines or primary tissues. In addition, a detailed explanation of the profiling system components has been included to aid in the understanding of its function, guide further optimization and elaboration, and provide information for troubleshooting.

Section snippets

Permeabilizing the plasma membrane

While mitochondrial potential dyes are capable of penetrating the plasma membrane, many BH3 profiling components are incapable of diffusing through this membrane. The most important of these are the BH3 peptides necessary to deliver the defined input used to trigger the apoptotic machinery, and the succinate that is needed to facilitate ΔΨm. In its earliest form, BH3 profiling surmounted this issue by isolating heavy membranes at the cost of losing all surface markers and most other

Effects of buffer composition

It is the purpose of any buffer to provide an environment that remains sufficiently stable to eliminate it as a source of variance. While it is easy to take for granted, the alteration of only one of many components can result in different results. Buffers used for ΔΨm measurements usually contain a sugar, a pH buffer, salts, chelators, and a carbon source for the electron transport chain at a minimum [22], [23], [24], [25]. Understanding the tolerance of ΔΨm measurements to variation of these

Measurement of ΔΨm

The detection of mitochondrial outer membrane permeabilization (MOMP) is the endpoint for all forms of BH3 profiling. This can be accomplished by measuring the exit of intermembrane space proteins such as SMAC/DIABLO and cytochrome c, but detection of these proteins require the use of antibody staining, ELISA, or western blot which are not amenable to real time measurements or high throughput assays, and tend to be more expensive and time-consuming. While the loss of cytochrome c from the

Cell permeant peptides

The use of a number of small peptide tags has allowed for whole proteins to be drawn into cells without the addition of other agents to facilitate their import. TAT, antennepedia, and poly-arginine are the best known sequences, and all three are cationic peptides that associate with the cell membrane prior to import. In some cases, this import can be swift and very effective.

In the context of BH3 profiling, we have observed import to be slow and its effects do not correlate well with the

Plate-based profiling

Current techniques for BH3 profiling are capable of handling both homogenous and heterogeneous cell samples. The choice of which techniques to use depends primarily on what sort of population is to be analyzed. In the case of homogeneous samples such as sorted cells, cells isolated from selection columns, or controlled cell lines, a plate-based approach (Fig. 5A) provides the most information and greatest throughput for the lowest expense. In general, if there is only one population to analyze

Conclusion

BH3 profiling is a phenotypic assay that can provide information about how dependent a cell is on individual anti-apoptotic family proteins. We have used this application to identify cells that are dependent on MCL-1 or BCL-2. The latter finding is of particular interest, as it may have use in selecting patients best suited to receive innovative BCL-2 antagonists like ABT-263 and ABT-199.

More recently we have been focusing on using BH3 to measure how close a cell is to the threshold of

Acknowledgements

The authors gratefully acknowledge support from the following sources: NIH grants F31 CA150562, P01 CA139980, and R01 CA129974. A.L. is a Leukemia and Lymphoma Society Scholar. A.L. was a cofounder and formerly served on the scientific advisory board of Eutropics Pharmaceuticals, which has a license for BH3 profiling.

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