title: Visualization of caspase-3-like activity in cells using a genetically encoded fluorescent biosensor activated by protein cleavage.
abstract: Cytosolic caspase-3-like proteases, such as caspase-3 and caspase-7, have a central role in mediating the progress of apoptosis. Here to conveniently monitor caspase-3-like activity in the multicellular environment, we have developed genetically encoded switch-on fluorescence-base indicators that are cyclized chimeras containing a caspase-3 cleavage site as a switch. When cleaved by caspase-3-like proteases, the non-fluorescent indicator rapidly becomes fluorescent, and thus detects in real-time the activation of such caspases. We generate cultured cells constitutively expressing these chimeras, and all the healthy cells are non-fluorescent. When these cells are exposed to apoptotic stimuli, dead cells show strong fluorescence depending on caspase activation. With these tools, we monitor in real-time caspase-3-like activity in each cell under various conditions, and show for the first time that the environment of cancer cells affects their sensitivity to chemotherapeutic drugs in a modified soft agar assay. These biosensors should enable better understanding of the biological relevance of caspase-3-like proteases.
authors.affiliation: Laboratory of Cancer Cell Biology, Key Laboratory of Breast Cancer Prevention and Therapy, Tianjin Medical University Cancer Institute and Hospital, Tianjin, China.
authors.forename: Jiao
authors.initials: J
authors.lastname: Zhang
authors.forename: Xin
authors.initials: X
authors.lastname: Wang
authors.forename: Wenjing
authors.initials: W
authors.lastname: Cui
authors.forename: Wenwen
authors.initials: W
authors.lastname: Wang
authors.forename: Huamei
authors.initials: H
authors.lastname: Zhang
authors.forename: Lu
authors.initials: L
authors.lastname: Liu
authors.forename: Zicheng
authors.initials: Z
authors.lastname: Zhang
authors.forename: Zheng
authors.initials: Z
authors.lastname: Li
authors.forename: Guoguang
authors.initials: G
authors.lastname: Ying
authors.forename: Ning
authors.initials: N
authors.lastname: Zhang
authors.forename: Binghui
authors.initials: B
authors.lastname: Li
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D015374:Biosensing Techniques / Q000379:methods*; D020169:Caspases / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D005453:Fluorescence; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D008856:Microscopy, Fluorescence; D057054:Molecular Imaging; D015335:Molecular Probes / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D015335:Molecular Probes; D014409:Tumor Necrosis Factor-alpha; D020169:Caspases