Interactions of pro-apoptotic BH3 proteins with anti-apoptotic Bcl-2 family proteins measured in live MCF-7 cells using FLIM FRET

  • Liu Q
  • Leber B
  • Andrews D
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Abstract

I ncreased interactions between pro-apoptotic BH3-only proteins and anti-apoptotic Bcl-2 family proteins at mitochondria result in tumor initia-tion, progression and resistance to tradi-tional chemotherapy. Drugs that mimic the BH3 region are expected to release BH3-only proteins from anti-apoptotic proteins, inducing apoptosis in some cancer cells and sensitizing others to che-motherapy. Recently, we applied fluores-cence lifetime imaging microscopy and fluorescence resonance energy transfer to measure protein:protein interactions for the Bcl-2 family of proteins in live MCF-7 cells using fluorescent fusion proteins. While the BH3-proteins bound to Bcl-XL and Bcl-2, the BH3 mimetic ABT-737 inhibited binding of only Bad and tBid, but not Bim. We have extended our studies by investigating ABT-263, a clinical drug based on ABT-737. We show that the inhibitory effects and pat-tern of the two drugs are comparable for both Bcl-XL and Bcl-2. Furthermore, we show that mutation of a conserved residue in the BH3 region in Bad and tBid disrupted their interactions with Bcl-XL and Bcl-2, while the correspond-ing BimEL mutant showed no decrease in binding to these anti-apoptotic pro-teins. Therefore, in MCF-7 cells, Bim has unique binding properties compared to other BH3-only proteins that resist displacement from Bcl-XL and Bcl-2 by BH3 mimetics. Apoptosis is a common mechanism used by multicellular organisms to remove cells that have damaged regulatory pathways controlling cellular proliferation and homeostatsis. The Bcl-2 family of proteins integrates various cellular signals and controls mito-chondrial outer membrane permeabiliza-tion, an event widely believed to commit cells to apoptosis. 1 Anti-apoptotic proteins, including Bcl-XL, Bcl-2 and Mcl-1, inhibit the executer proteins BAX and BAK that, once activated, oligomerize and perme-ablize mitochondria, and the BH3-only proteins that directly (Bid and Bim) or indirectly (Bad) activate BAX and BAK. 2 One of the key common features of can-cer cells is the failure of apoptosis that is often caused by the overexpression of anti-apoptotic proteins that neutralize the pro-apoptotic signaling generated by aber-rant growth control. 3,4 Thus, it is expected that inhibiting anti-apoptotic proteins may provide an effective way to selec-tively kill cancer cells or sensitize them to chemotherapy. Based on this concept many anticancer drugs are under development; however, ABT-737 and ABT-263, inhibitors of both Bcl-XL and Bcl-2, are the most successful ones. Designed to mimic the BH3-only protein Bad, ABT-737 has nanomolar affinities for Bcl-XL and Bcl-2 when sol-uble fragments are used as binding targets and displaces BH3-only proteins from the binding pocket of Bcl-XL. 5,6

Author-supplied keywords

  • ABT-263
  • ABT-737
  • BH3-only proteins
  • Bcl-2
  • Bcl-XL
  • Bim
  • Live MCF-7 cells
  • Mitochondria
  • Protein-protein interactions

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Authors

  • Qian Liu

  • Brian Leber

  • David W. Andrews

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