Accessible Unlicensed Requires Authentication Published by De Gruyter February 23, 2016

iBH3: simple, fixable BH3 profiling to determine apoptotic priming in primary tissue by flow cytometry

Jeremy Ryan ORCID logo, Joan Montero, James Rocco and Anthony Letai
From the journal Biological Chemistry

Abstract

Dysregulation of the mitochondrial pathway of apoptosis, controlled by the BCL-2 family of proteins, leads to disease states including cancer. Rapid analysis of a cell’s dependency on the BCL-2 family of proteins is hindered by the complex interactions of more than a dozen proteins. Transcript or even protein levels are therefore generally insufficient to predict a cell’s response to perturbations like chemotherapy. Previously, we developed the JC-1 BH3 method to provide a same day functional assay to assess a cell’s propensity to undergo apoptosis and demonstrated its utility in predicting response to chemotherapy. We have now improved upon these methods to create a robust assay amenable to high throughput platforms using cytochrome c retention in formaldehyde fixed cells to remove the time sensitivity of JC-1 potential measurements. BH3 profiling by intracellular staining (iBH3) is suitable for 96- and 384-well formats, and can be used to directly screen candidate BH3-mimetic compounds for activity. When used as the final component of dynamic BH3 profiling (DBP), which uses a drug pretreatment prior to iBH3 to assess the change in profile due to treatment, it can predict the response of cells to chemotherapy days before they show signs of death.

References

Aragon, J.J., Feliu, J.E., Frenkel, R.A., and Sols, A. (1980). Permeabilization of animal cells for kinetic studies of intracellular enzymes: in situ behavior of the glycolytic enzymes of erythrocytes. Proc. Natl. Acad. Sci. USA 77, 6324–6328.Search in Google Scholar

Brunelle, J.K. and Letai, A. (2009). Control of mitochondrial apoptosis by the Bcl-2 family. J. Cell Sci. 122, 437–441.Search in Google Scholar

Cassany, A. and Gerace, L. (2009). Reconstitution of nuclear import in permeabilized cells. Methods Mol. Biol. 464, 181–205.Search in Google Scholar

Certo, M., Del Gaizo Moore, V., Nishino, M., Wei, G., Korsmeyer, S., Armstrong, S.A., and Letai, A. (2006). Mitochondria primed by death signals determine cellular addiction to antiapoptotic BCL-2 family members. Cancer Cell 9, 351–365.Search in Google Scholar

Fiskum, G., Craig, S.W., Decker, G.L., and Lehninger, A.L. (1980). The cytoskeleton of digitonin-treated rat hepatocytes. Proc. Natl. Acad. Sci. USA 77, 3430–3434.Search in Google Scholar

Foight, G.W., Ryan, J.A., Gulla, S.V., Letai, A., and Keating, A.E. (2014). Designed BH3 peptides with high affinity and specificity for targeting Mcl-1 in cells. ACS Chem. Biol. 9, 1962–1968.Search in Google Scholar

Liu, J., Xiao, N., and DeFranco, D.B. (1999). Use of digitonin-permeabilized cells in studies of steroid receptor subnuclear trafficking. Methods 19, 403–409.Search in Google Scholar

Mason, K.D., Carpinelli, M.R., Fletcher, J.I., Collinge, J.E., Hilton, A.A., Ellis, S., Kelly, P.N., Ekert, P.G., Metcalf, D., Roberts, A.W., et al. (2007). Programmed anuclear cell death delimits platelet life span. Cell 128, 1173–1186.Search in Google Scholar

Montero, J., Sarosiek, K.A., DeAngelo, J.D., Maertens, O., Ryan, J., Ercan, D., Piao, H., Horowitz, N.S., Berkowitz, R.S., Matulonis, U., et al. (2015). Drug-induced death signaling strategy rapidly predicts cancer response to chemotherapy. Cell 160, 977–989.Search in Google Scholar

Ni Chonghaile, T., Sarosiek, K.A., Vo, T.T., Ryan, J.A., Tammareddi, A., Moore, V. del G., Deng, J., Anderson, K.C., Richardson, P., et al. (2011). Pretreatment mitochondrial priming correlates with clinical response to cytotoxic chemotherapy. Science 334, 1129–1133.Search in Google Scholar

Ryan, J. and Letai, A. (2013). BH3 profiling in whole cells by fluorimeter or FACS. Methods 61, 156–164.Search in Google Scholar

Ryan, J.A., Brunelle, J.K., and Letai, A. (2010). Heightened mitochondrial priming is the basis for apoptotic hypersensitivity of CD4+ CD8+ thymocytes. Proc. Natl. Acad. Sci. USA 107, 12895–12900.Search in Google Scholar

Vo, T.T., Ryan, J., Carrasco, R., Neuberg, D., Rossi, D.J., Stone, R.M., Deangelo, D.J., Frattini, M.G., and Letai, A. (2012). Relative mitochondrial priming of myeloblasts and normal HSCs determines chemotherapeutic success in AML. Cell 151, 344–355.Search in Google Scholar

Zhang, H., Nimmer, P.M., Tahir, S.K., Chen, J., Fryer, R.M., Hahn, K.R., Iciek, L.A., Morgan, S.J., Nasarre, M.C., Nelson, R., et al. (2007). Bcl-2 family proteins are essential for platelet survival. Cell Death Differ. 14, 943–951.Search in Google Scholar

Received: 2016-1-11
Accepted: 2016-2-15
Published Online: 2016-2-23
Published in Print: 2016-7-1

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