The use of a stably expressed FRET biosensor for determining the potency of cancer drugs

William P Bozza; Xu Di; Kazuyo Takeda; Leslie A Rivera Rosado; Sarah Pariser; Baolin Zhang

doi:10.1371/journal.pone.0107010

The use of a stably expressed FRET biosensor for determining the potency of cancer drugs

PLoS One. 2014 Sep 4;9(9):e107010. doi: 10.1371/journal.pone.0107010. eCollection 2014.

Authors

William P Bozza¹, Xu Di¹, Kazuyo Takeda², Leslie A Rivera Rosado¹, Sarah Pariser³, Baolin Zhang¹

Affiliations

¹ 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.
² Microscopy and Imaging Core Facility, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, United States of America.
³ 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.

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.

Publication types

Research Support, U.S. Gov't, P.H.S.

MeSH terms

Amino Acid Sequence
Antineoplastic Agents / pharmacology*
Apoptosis / drug effects*
Bacterial Proteins / genetics
Bacterial Proteins / metabolism
Biological Assay*
Biosensing Techniques*
Caspase 3 / genetics
Caspase 3 / metabolism
Caspase 8 / genetics
Caspase 8 / metabolism
Cell Line, Tumor
Epithelial Cells / drug effects*
Epithelial Cells / metabolism
Epithelial Cells / pathology
Fluorescence Resonance Energy Transfer
Gene Expression Regulation
Genes, Reporter
Green Fluorescent Proteins / genetics
Green Fluorescent Proteins / metabolism
Humans
Luminescent Proteins / genetics
Luminescent Proteins / metabolism
Mammary Glands, Human / drug effects
Mammary Glands, Human / metabolism
Mammary Glands, Human / pathology
Molecular Sequence Data
Signal Transduction

Substances

Antineoplastic Agents
Bacterial Proteins
Cyan Fluorescent Protein
Luminescent Proteins
yellow fluorescent protein, Bacteria
Green Fluorescent Proteins
Caspase 3
Caspase 8

Grants and funding

This work was supported by the Food and Drug Administration, Center for Drug Evaluation and Research, Critical Path Initiative fund; and the Food and Drug Administration, Center for Drug Evaluation and Research, Medical Countermeasure Initiative fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.