title: Design and development of a whole-cell luminescent biosensor for detection of early-stage of apoptosis.
abstract: We present here a novel whole-cell biosensor to detect early-stages of apoptosis based on Apaf-1 oligomerization and apoptosome formation using the split luciferase strategy. The amino-fragment (1-416 amino acids) and carboxy-fragment (395-550 amino acids) of firefly luciferase were fused to amino-terminal of Apaf-1. The cotransfected HEK cells were then treated with doxorubicin for induction of apoptosis. The performance of our biosensor for monitoring of programmed cell death over 24h was investigated by measuring bioluminescence activities. We observed a significant increase (≈ 15 fold) in luminescence signal compared to control cells 4h after apoptosis induction. It reached a maximum activity over 10h (≈ 155 fold). Moreover, juxtapositioning of Apaf-1 monomer and apoptosome formation occur about 5h earlier than the appearance of significant caspase3/7 activity upon induction of apoptosis by doxorubicin. The time-response curve of split luciferase shows a sigmoidal pattern which indicates cooperativity in oligomerization of Apaf-1 upon binding of cytochrome c. This biosensor can be used as a new platform, based on the protein fragment complementation strategy for assessing potential chemotherapeutic drugs as well as a sensitive and dynamic system in the time- and dose-dependent studies of apoptosis.
authors: Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
authors: Masoud
authors: M
authors: Torkzadeh-Mahani
authors: Farangis
authors: F
authors: Ataei
authors: Maryam
authors: M
authors: Nikkhah
authors: Saman
authors: S
authors: Hosseinkhani
mesh_terms: D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D017209:Apoptosis; D053477:Apoptotic Protease-Activating Factor 1 / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D015374:Biosensing Techniques / Q000295:instrumentation*; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D004317:Doxorubicin / Q000494:pharmacology; D004867:Equipment Design; D048888:Fireflies / Q000201:enzymology; D057809:HEK293 Cells; D006801:Humans; D049409:Luciferases, Firefly / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D049408:Luminescent Agents / Q000032:analysis / Q000378:metabolism; D008163:Luminescent Measurements / Q000295:instrumentation*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D000903:Antibiotics, Antineoplastic; D053477:Apoptotic Protease-Activating Factor 1; D049408:Luminescent Agents; D011993:Recombinant Fusion Proteins; D004317:Doxorubicin; D049409:Luciferases, Firefly; D053148:Caspase 3; D053179:Caspase 7