Molecular analysis of functional redundancy among anti-apoptotic Bcl-2 proteins and its role in cancer cell survival

Exp Cell Res. 2014 Apr 1;322(2):415-24. doi: 10.1016/j.yexcr.2014.02.010. Epub 2014 Feb 17.

Abstract

Bcl-2 family proteins act as essential regulators and mediators of intrinsic apoptosis. Several lines of evidence suggest that the anti-apoptotic members of the family, including Bcl-2, Bcl-xL and Mcl-1, exhibit functional redundancy. However, the current evidence is largely indirect, and based mainly on pharmacological data using small-molecule inhibitors. In order to study compensation and redundancy of anti-apoptotic Bcl-2 proteins at the molecular level, we used a combined knockdown/overexpression strategy to essentially replace the function of one member with another. The results show that HeLa cells are strictly dependent on Mcl-1 for survival and correspondingly refractory to the Bcl-2/Bcl-xL inhibitor ABT-263, and remain resistant to ABT-263 in the context of Bcl-xL overexpression because endogenous Mcl-1 continues to provide the primary guardian role. However, if Mcl-1 is knocked down in the context of Bcl-xL overexpression, the cells become Bcl-xL-dependent and sensitive to ABT-263. We also show that Bcl-xL compensates for loss of Mcl-1 by sequestration of two key pro-apoptotic Bcl-2 family members, Bak and Bim, normally bound to Mcl-1, and that Bim is essential for cell death induced by Mcl-1 knockdown. To our knowledge, this is the first example where cell death induced by loss of Mcl-1 was rescued by the silencing of a single BH3-only Bcl-2 family member. In colon carcinoma cell lines, Bcl-xL and Mcl-1 also play compensatory roles, and Mcl-1 knockdown sensitizes cells to ABT-263. The results, obtained employing a novel strategy of combining knockdown and overexpression, provide unique molecular insight into the mechanisms of compensation by pro-survival Bcl-2 family proteins.

Keywords: ABT-263; Apoptosis; Bcl-2; Bcl-xL; Mcl-1.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Aniline Compounds / pharmacology
  • Antineoplastic Agents / pharmacology
  • Apoptosis Regulatory Proteins / metabolism*
  • Bcl-2-Like Protein 11
  • Blotting, Western
  • Caspase 3 / metabolism
  • Cell Survival / drug effects*
  • HeLa Cells
  • Humans
  • Immunoprecipitation
  • Membrane Proteins / metabolism
  • Myeloid Cell Leukemia Sequence 1 Protein / antagonists & inhibitors
  • Myeloid Cell Leukemia Sequence 1 Protein / metabolism*
  • Proto-Oncogene Proteins / metabolism
  • Proto-Oncogene Proteins c-bcl-2 / metabolism*
  • Signal Transduction / drug effects
  • Sulfonamides / pharmacology
  • bcl-2 Homologous Antagonist-Killer Protein / metabolism
  • bcl-X Protein / metabolism*

Substances

  • Aniline Compounds
  • Antineoplastic Agents
  • Apoptosis Regulatory Proteins
  • BAK1 protein, human
  • BCL2L1 protein, human
  • BCL2L11 protein, human
  • Bcl-2-Like Protein 11
  • MCL1 protein, human
  • Membrane Proteins
  • Myeloid Cell Leukemia Sequence 1 Protein
  • Proto-Oncogene Proteins
  • Proto-Oncogene Proteins c-bcl-2
  • Sulfonamides
  • bcl-2 Homologous Antagonist-Killer Protein
  • bcl-X Protein
  • Caspase 3
  • navitoclax