title: The use of a stably expressed FRET biosensor for determining the potency of cancer drugs.
abstract: Many cancer drugs are intended to kill cancer cells by inducing apoptosis. However, the potency assays used for measuring the bioactivity of these products are generally cell viability assays which do not distinguish between cell death and growth inhibition. Here we describe a cell-based fluorescence resonance energy transfer (FRET) biosensor designed to measure the bioactivity of apoptosis inducing cancer drugs. The biosensor contains cyan fluorescent protein (CFP) linked via caspase 3 and caspase 8 specific cleavage recognition sequences to yellow fluorescent protein (YFP). Upon caspase activation, as in the case of apoptosis induction, the linker is cleaved abolishing the cellular FRET signal. This assay closely reflects the mechanism of action of cancer drugs, in killing cancer cells and therefore can function as a potency test for different cancer drugs. We rigorously demonstrate this through characterization of a class of proteins targeting the death receptors. The one-step assay appears to be superior to other apoptosis-based assays because of its simplicity, convenience, and robustness.
authors.affiliation: Division of Therapeutic Proteins, Office of Biotechnology Products, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, United States of America.
authors.forename: William P
authors.initials: WP
authors.lastname: Bozza
authors.affiliation: Division of Therapeutic Proteins, Office of Biotechnology Products, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, United States of America.
authors.forename: Xu
authors.initials: X
authors.lastname: Di
authors.affiliation: Microscopy and Imaging Core Facility, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, United States of America.
authors.forename: Kazuyo
authors.initials: K
authors.lastname: Takeda
authors.affiliation: Division of Therapeutic Proteins, Office of Biotechnology Products, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, United States of America.
authors.forename: Leslie A
authors.initials: LA
authors.lastname: Rivera Rosado
authors.affiliation: Division of Therapeutic Proteins, Office of Biotechnology Products, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, United States of America; Brown University, Providence, Rode Island, United States of America.
authors.forename: Sarah
authors.initials: S
authors.lastname: Pariser
authors.affiliation: Division of Therapeutic Proteins, Office of Biotechnology Products, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, United States of America.
authors.forename: Baolin
authors.initials: B
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001681:Biological Assay; D015374:Biosensing Techniques; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D053181:Caspase 8 / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D031541:Fluorescence Resonance Energy Transfer; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D042361:Mammary Glands, Human / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D008969:Molecular Sequence Data; D015398:Signal Transduction
chemical_list: D000970:Antineoplastic Agents; D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D053148:Caspase 3; D053181:Caspase 8