title: Molecular analysis of functional redundancy among anti-apoptotic Bcl-2 proteins and its role in cancer cell survival.
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.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.
authors.forename: Joshua M
authors.initials: JM
authors.lastname: Eichhorn
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Alford
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.
authors.forename: Nandini
authors.initials: N
authors.lastname: Sakurikar
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA. Electronic address: chamberstimothyc@uams.edu.
authors.forename: Timothy C
authors.initials: TC
authors.lastname: Chambers
mesh_terms: D000814:Aniline Compounds / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism*; D000072224:Bcl-2-Like Protein 11; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects*; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D008565:Membrane Proteins / Q000378:metabolism; D064549:Myeloid Cell Leukemia Sequence 1 Protein / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013449:Sulfonamides / Q000494:pharmacology; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000378:metabolism; D051020:bcl-X Protein / Q000378:metabolism*
chemical_list: D000814:Aniline Compounds; D000970:Antineoplastic Agents; D051017:Apoptosis Regulatory Proteins; C494772:BAK1 protein, human; C494811:BCL2L1 protein, human; C000606775:BCL2L11 protein, human; D000072224:Bcl-2-Like Protein 11; C579095:MCL1 protein, human; D008565:Membrane Proteins; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D013449:Sulfonamides; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051020:bcl-X Protein; D053148:Caspase 3; C528561:navitoclax
keywords: ABT-263; Apoptosis; Bcl-2; Bcl-xL; Mcl-1