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1.
Biol Res ; 42(2): 249-60, 2009.
Article in English | MEDLINE | ID: mdl-19746271

ABSTRACT

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein that mediates both NADH-oxidizing and caspase-independent apoptosis. Further, the proapoptotic activity of AIF is located in the C-terminus of AIF, although the precise minimum sequence responsible for apoptosis induction remains to be investigated. In the present study, we generated two truncated AIFs, AIFDelta1-480-FLAG, which is a FLAG-tagged C-terminal peptide comprising amino acids from 481 to 613, and AIF360-480 containing amino acids from 360 to 480 of AIF. We used confocal microscopy to demonstrate that both the truncated proteins are expressed and located in the cytoplasm of transfected cells. AIFDelta1-480 but not AIF360-480 induces apoptosis in transfected cells. We also found that the expression of AIFDelta1-480 could initiate the release of cytochrome c from the mitochondria. The suppression of caspase-9 via siRNA blocked the proapoptotic activity of AIFDelta1-480. Therefore, AIFDelta1-480 is sufficient for inducing caspase-9-dependent apoptotic signaling, probably by promoting the release of cytochrome c. At last, we generated a chimeric immuno-AIFDelta1-480 protein, which comprised an HER2 antibody, a Pseudomonas exotoxin A translocation domain and AIFDelta1-480. Human Jurkat cells transfected with the immuno-AIFDeltal-480 gene could express and secrete the chimeric protein, which selectively recognize and kill HER2-overexpressing tumor cells. Our study demonstrates the feasibility of the immuno-AIFDeltal-480 gene as a novel approach to treating HER2-overexpressing cancers.


Subject(s)
Alcohol Oxidoreductases/drug effects , Apoptosis Inducing Factor/pharmacology , Apoptosis/drug effects , DNA, Complementary/drug effects , DNA-Binding Proteins/drug effects , Alcohol Oxidoreductases/genetics , Alcohol Oxidoreductases/metabolism , Apoptosis/genetics , Apoptosis Inducing Factor/genetics , Apoptosis Inducing Factor/metabolism , Blotting, Western , DNA, Complementary/genetics , DNA, Complementary/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Fluorescent Antibody Technique, Indirect , Humans , Jurkat Cells , Microscopy, Confocal , Reverse Transcriptase Polymerase Chain Reaction , Transfection
2.
Biol. Res ; 42(2): 249-260, 2009. ilus, tab
Article in English | LILACS | ID: lil-524896

ABSTRACT

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein that mediates both NADH-oxidizing and caspase-independent apoptosis. Further, the proapoptotic activity of AIF is located in the C-terminus of AIF, although the precise minimum sequence responsible for apoptosis induction remains to be investigated. In the present study, we generated two truncated AIFs, AIFΔ1-480-FLAG, which is a FLAG-tagged C-terminal peptide comprising amino acids from 481 to 613, and AIF360-480 containing amino acids from 360 to 480 of AIF. We used confocal microscopy to demonstrate that both the truncated proteins are expressed and located in the cytoplasm of transfected cells. AIFΔ1-480 but not AIF360-480 induces apoptosis in transfected cells. We also found that the expression of AIFΔ1-480 could initiate the release of cytochrome c from the mitochondria. The suppression of caspase-9 via siRNA blocked the proapoptotic activity of AIFΔ1-480. Therefore, AIFΔ 1-480 is sufficient for inducing caspase-9-dependent apoptotic signaling, probably by promoting the release of cytochrome c. At last, we generated a chimeric immuno-AIFΔ 1-480 protein, which comprised an HER2 antibody, a Pseudomonas exotoxin A translocation domain and AIFΔ 1-480. Human Jurkat cells transfected with the immuno-AIFΔl-480 gene could express and secrete the chimeric protein, which selectively recognize and kill HER2-overexpressing tumor cells. Our study demonstrates the feasibility of the immuno-AIFΔl-480 gene as a novel approach to treating HER2-overexpressing cancers.


Subject(s)
Humans , Alcohol Oxidoreductases/drug effects , Apoptosis Inducing Factor/pharmacology , Apoptosis/drug effects , DNA, Complementary/drug effects , DNA-Binding Proteins/drug effects , Alcohol Oxidoreductases/genetics , Alcohol Oxidoreductases/metabolism , Apoptosis Inducing Factor/genetics , Apoptosis Inducing Factor/metabolism , Apoptosis/genetics , Blotting, Western , DNA, Complementary/genetics , DNA, Complementary/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Fluorescent Antibody Technique, Indirect , Jurkat Cells , Microscopy, Confocal , Reverse Transcriptase Polymerase Chain Reaction , Transfection
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