Bozza William P, Di Xu, Takeda Kazuyo, Rivera Rosado Leslie A, Pariser Sarah, Zhang Baolin
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.
Microscopy and Imaging Core Facility, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, Maryland, United States of America.
PLoS One. 2014 Sep 4;9(9):e107010. doi: 10.1371/journal.pone.0107010. eCollection 2014.
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.
许多抗癌药物旨在通过诱导细胞凋亡来杀死癌细胞。然而，用于测量这些产品生物活性的效力测定通常是细胞活力测定，它无法区分细胞死亡和生长抑制。在此，我们描述了一种基于细胞的荧光共振能量转移（FRET）生物传感器，旨在测量诱导细胞凋亡的抗癌药物的生物活性。该生物传感器包含通过半胱天冬酶3和半胱天冬酶8特异性切割识别序列与黄色荧光蛋白（YFP）相连的青色荧光蛋白（CFP）。在半胱天冬酶激活时，如在诱导细胞凋亡的情况下，连接子被切割，消除了细胞内的FRET信号。该测定法密切反映了抗癌药物杀死癌细胞的作用机制，因此可作为不同抗癌药物的效力测试。我们通过对一类靶向死亡受体的蛋白质进行表征，严格证明了这一点。由于其简单、方便和稳健性，这种一步测定法似乎优于其他基于细胞凋亡的测定法。
