ABSTRACT
B cell lymphoma 2 (Bcl-2) homology domain 3 (BH3)-only proteins of the Bcl-2 family are important functional adaptors that link cell death signals to the activation of Bax and/or Bak. The BH3-only protein Nbk/Bik induces cell death via an entirely Bax-dependent/Bak-independent mechanism. In contrast, cell death induced by the short splice variant of Bcl-x depends on Bak but not Bax. This indicates that Bak is functional but fails to become activated by Nbk. Here, we show that binding of myeloid cell leukemia 1 (Mcl-1) to Bak persists after Nbk expression and inhibits Nbk-induced apoptosis in Bax-deficient cells. In contrast, the BH3-only protein Puma disrupts Mcl-1-Bak interaction and triggers cell death via both Bax and Bak. Targeted knockdown of Mcl-1 overcomes inhibition of Bak and allows for Bak activation by Nbk. Thus, Nbk is held in check by Mcl-1 that interferes with activation of Bak. The finding that different BH3-only proteins rely specifically on Bax, Bak, or both has important implications for the design of anticancer drugs targeting Bcl-2.
Subject(s)
Apoptosis Regulatory Proteins/metabolism , Apoptosis , Membrane Proteins/metabolism , Neoplasm Proteins/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , bcl-2 Homologous Antagonist-Killer Protein/metabolism , bcl-2-Associated X Protein/metabolism , Adenoviridae/genetics , Apoptosis/genetics , Apoptosis/physiology , Benzimidazoles , Carbocyanines , Cell Line , Cell Line, Tumor , Cell Membrane Permeability , Cytochromes c/metabolism , Fluorescent Dyes , HCT116 Cells , Humans , Kidney/cytology , Male , Mitochondria/physiology , Mitochondrial Proteins , Models, Biological , Myeloid Cell Leukemia Sequence 1 Protein , Prostatic Neoplasms/pathology , Transgenes , bcl-2 Homologous Antagonist-Killer Protein/genetics , bcl-2-Associated X Protein/geneticsABSTRACT
The use of shRNA for knockdown of gene expression is a powerful method. In addition to transient transfection of RNA oligonucleotides, various DNA-based vectors that express short hairpin RNAs have been successfully used for efficient depletion of gene products. Replication-defective retrovirus and adenovirus (Ad) vectors have also gained wide usage. The extension of shRNA technology to replication-competent Ad would be desirable to investigate the role of various cellular genes in Ad replication. This approach is hampered because the effect of shRNA is neutralized by the Ad VA-RNA that is expressed at late stages after infection, and the infected cells are killed prior to significant depletion of some long-lived target gene products. We have constructed replication-competent Ad vectors for the depletion of the pro-apoptotic proteins BAX and BAK. We have modified a replication-defective Ad multivalent shRNA expression vector developed by Welgen, Inc. In our vector design, the multivalent shRNA expression cassette is contained in the E1B region. Additionally, we have incorporated a temperature-sensitive mutation in the viral DBP gene (ts125). The use of this vector has resulted in efficient depletion of critical cellular apoptotic modulators, BAX and BAK. This vector may be useful to study the role of various cellular genes in Ad-induced apoptosis and viral replication.
